Gamma-band oscillations are implicated in modulation of attention, integration of sensory information and flexible communication among anatomically connected brain areas. How networks become entrained is incompletely understood. Specifically, it is unclear how the spectral and temporal characteristics of network oscillations can be altered on rapid timescales needed for efficient communication. We use closed-loop optogenetic modulation of principal cell excitability in mouse hippocampal slices to interrogate the dynamical properties of hippocampal oscillations. Gamma frequency and amplitude can be modulated bi-directionally, and dissociated, by phase-advancing or delaying optogenetic feedback to pyramidal cells. Closed-loop modulation alters the synchrony rather than average frequency of action potentials, in principle avoiding disruption of population rate-coding of information. Modulation of phasic excitatory currents in principal neurons is sufficient to manipulate oscillations, suggesting that feed-forward excitation of pyramidal cells has an important role in determining oscillatory dynamics and the ability of networks to couple with one another. Gamma-band (approximately 30 to 120 Hz) oscillations have been implicated in the modulation of attention and perception, in action initiation, spatial navigation and memory encoding, and have also been proposed to underlie flexible information routing among anatomically connected regions . CentralAlthough the cellular mechanisms underlying gamma oscillations have been extensively studied , there rFurther insight into the dynamical mechanisms of synchronization between oscillating networks comes from examining the phase response curve (PRC) of the network oscillation, defined as the phase advance or delay produced by a transient stimulation, as a function of the instantaneous phase at which the stimulus is delivered. The finding that gamma in an in vitro hippocampal slice preparation shows a biphasic PRC  is consiThe present study investigates the dynamical properties of gamma oscillations by using closed-loop optogenetics to create an artificial feedback loop between the oscillatory network activity (as assessed by the LFP) and excitatory input to the principal cell population. Specifically, we delivered analogue-modulated excitation whose strength was a function of the instantaneous phase and amplitude of the oscillation. This approach is quite distinct from previous closed-loop applications of optogenetics , which hCamk2a promoter to bias expression to excitatory neurons. The local field potential (LFP) was recorded in the CA1 pyramidal cell layer in acute hippocampal slices. A slowly increasing ramp of light (peak wavelength 590 nm) was delivered via a light-emitting diode (LED) coupled to the epifluorescence port of an upright microscope, eliciting a gamma oscillation , where k1 and k2 are positive or negative constants. These operations were implemented with a field-programmable gate array (FPGA) and applied for a defined duration  during the ramp. This yielded a change in the spectral properties of the oscillation, which lasted for the duration of the closed-loop feedback  previously reported . In thatAlthough the shape of the PRC is consistent with the changes in gamma frequency achieved with closed-loop modulation at different LED-LFP phase differences, on its own it says nothing about changes in power. Power was maximally decreased with a phase advance of the LED command over the LFP around 90°, whilst it was maximally increased with a phase delay around 90° . The relA deeper understanding of the characteristic changes in frequency and power of the LFP with different LED-LFP phase differences requires an insight into how neurons spike and ultimately how membrane currents respond to the fluctuations in optogenetic drive. We therefore performed single-cell recordings in parallel with the LFP recordings.Although the average illumination intensity was not altered during the gamma clamp, for certain LED-LFP phase relationships gamma power increased or decreased robustly. Inhibitory currents in principal neurons, rather than spikes or excitatory currents, have previously been shown to be the main determinant of the LFP , suggest−7, n = 7, Hotellier test – Increases in oscillatory frequency were accompanied by a phase advance of pyramidal cell firing relative to the LFP , and the other pipette to record the LFP. We then measured the inward current at each phase of the gamma oscillation, as defined by the LFP, and repeated this over consecutive cycles to obtain an average time-course . The mean phase and amplitude of outward inhibitory currents were calculated in a similar way, by subtracting the minimal current from the circular average of the outward GABA current . In cont current . Changes current , there w current . Alignin current . For the current , left. IThe present study shows that closed-loop optogenetic manipulation of principal cells allows predictable, bidirectional and dissociable changes in the power and frequency of gamma oscillations. We observed a broad consistency between the frequency manipulation achieved with closed-loop optogenetic feedback and that predicted from the phase response behavior previously observed with intermittent optogenetic stimuli . OptogenPrevious studies have stressed the importance of fast-spiking parvalbumin-positive (PV+) interneurons in gamma  but see). PV+ baClosed-loop manipulations have been applied previously in the context of network oscillations, using either electrical and optogenetic stimuli delivered at specific phases of theta or gamma oscillations, in order to probe the mechanisms of long-term plasticity induction  or sharpWe have focused on gamma because a local circuit is sufficient to generate the oscillation, and we have previously shown that the phase response behaviour of hippocampal gamma is well described by a simple dynamical model . The cirThe ability to alter the amplitude and frequency of gamma suggests a versatile tool to test the roles of gamma in information routing and other high-level brain functions, both in health and in disease states such as schizophrenia . HithertClosed-loop stimulation, in which signals recorded from a network are used in real time to bias its state, in principle provides an alternative way of manipulating network oscillations, and has been used to interfere with pathological rhythms in models of Parkinson’s disease , to suppExtrapolating from in vitro gamma to the brain in situ presents several technical challenges, including the need for optical fibers to illuminate the tissue and the potential for photoelectrical artifacts. Moreover, oscillations are generally less prominent because the current generators from multiple oscillating and non-oscillating populations overlap, complicating the evaluation of phase and frequency. Nevertheless, the present study identifies some general principles to guide attempts to achieve bidirectional and dissociable modulation of oscillatory frequency and power in vivo. This should allow a definitive test of the causal role of gamma in functions such as attention modulation and information routing .12 IU/ml) was injected at a rate of 100 nl/min into four sites in both hippocampi (injection volume: 300–500 nl per site). The antero-posterior injection coordinate was taken as 2/3 of the distance from bregma to lambda. The lateral coordinates were 3.0 mm from the midline, and the ventral coordinates were 3.5, 3.0, 2.5 and 2.0 mm from the surface of the skull.All procedures followed the Animals (Scientific Procedures) Act, 1986, and were reviewed by the UCL Institute of Neurology Animal Welfare and Ethical Review Body. P21 male C57 mice were anesthetized with isoflurane and placed in a stereotaxic frame (Kopf Instruments). A suspension of AAV5-CaMKIIa-C1V1(E122T/E162T)-TS-eYFP  were prepared at room temperature and then incubated at 37°C for 12 min in the same solution. They were subsequently stored at room temperature, in a solution containing (in mM): 126 NaCl, 3 KCl, 1.25 NaH2PO4, 2 MgSO4, 2 CaCl2, 24 NaHCO3, 10 glucose, shielded from light, before being transferred to the stage of an upright microscope (Olympus BX51WI or Scientifica SliceScope), where they were perfused on both sides with the same solution at 32° C. Expression of C1V1 in CA1 was verified by epifluorescence, and CA3 was ablated to focus on local gamma-generating mechanisms.Hippocampal slices were prepared at least 4 weeks later. Animals were sacrificed by pentobarbitone overdose and underwent transcardiac perfusion with an oxygenated solution containing (in mM): 92 N-methyl-D-glucamine-Cl, 2.5 KCl, 1.25 NaH2. Light ramps typically lasting 8 s were delivered every 30 – 45 s.Epifluorescence imaging and C1V1 stimulation were achieved with LEDs . The light source was coupled to the epifluorescence illuminator of the microscope, with a silver mirror in the place of a dichroic cube. Wide-field illumination was delivered via a 20x, 0.5 NA water immersion objective. The current delivered to the LED was kept in the linear input-output range, and the irradiance was <5 mW/mmk1LFP + k2dLFP/dt), stepping through different values of k in a pseudo-random order for successive trials. dLFP/dt was calculated as the difference between successive digitization values in the FPGA, averaged over successive 2 ms intervals to minimize high-frequency noise. The output of the FPGA/real-time controller was sent to the LED driver, and digitized in parallel with the LFP at 10 kHz on the data acquisition PC.LFPs were recorded in the CA1 pyramidal layer using patch pipettes filled with extracellular solution and a Multiclamp 700B amplifier (Molecular Devices), and band-pass filtered between 1 and 200 or 500 Hz. A linear LED ramp command was generated via a multifunction data acquisition card  and, together with the LFP, was digitized using a real-time controller  with a Xilinx Virtex-5 FPGA (cRIO-9133) operating at a loop rate of 10 kHz. The ramp was multiplied by : K-gluconate (145), NaCl (8), KOH-HEPES (10), EGTA (0.2), Mg-ATP (2) and Na 3 -GTP (0.3); pH 7.2; 290 mOsm. Phasic conductances  were estOff-line analysis was performed in LabVIEW  and R. Time-frequency spectrograms were calculated using a Morlet wavelet transform and are displayed as heat maps. Because the gamma oscillation was non-stationary, its frequency was estimated by calculating the short-term Fourier transform and then averaging the mean instantaneous frequency for successive overlapping intervals. The power of the oscillation was estimated in the same way, by averaging the power at the mean instantaneous frequency.Spikes were identified using threshold crossing. The instantaneous oscillation phase was estimated by passing a 200 ms segment of the LFP centered on the spike through a Hanning window, and then calculating its phase and frequency using the Extract Single Tone VI in LabVIEW.To estimate the phase relationship between spikes or membrane currents and the gamma oscillation, we first identified successive troughs of the LFP using the WA Multiscale Peak Detection VI in LabVIEW. Gamma cycles that deviated more than 20% from the modal period were rejected. The membrane current waveform between successive troughs was then expressed as a function of instantaneous phase and averaged over all accepted cycles in the interval. The minimal (least negative) inward current recorded at −70 mV was subtracted to yield the average phasic excitatory current waveform. To estimate the phasic inhibitory current waveform, the minimal outward current was subtracted whilst holding cells at 0 mV. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Michael Frank as the Senior Editor. The following individual involved in review of your submission has agreed to reveal his identity: Imre Vida (Reviewer #2). Two other reviewers remain anonymous.Thank you for submitting your article "Analog closed-loop optogenetic modulation of hippocampal pyramidal cells dissociates γ frequency and amplitude" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:Nicholson et al. used an optogenetic approach to examine the influence of phasic excitation of pyramidal cells (PCs) on the spectral and temporal dynamics of γ oscillations in acute slices of the hippocampal CA1 area. The authors demonstrate that phase-advancing or phase-delaying excitatory inputs onto PCs alter the synchrony but not the frequency of action potentials generated in CA- PCs. The authors recorded action potentials from pyramidal cells in the cell attached mode to examine how γ-clamp affects the timing and rate of action potentials generated by pyramidal cells. A decrease in oscillatory power was accompanied by a desynchronization of pyramidal cell activity whereas an increase in power was associated with an increase in the synchrony of PC discharges. The overall activity did not contribute to network synchrony. Increase in oscillatory frequency was associated with a phase advance of PC firing but in-phase modulation resulted in an increase in oscillatory power. Next, the authors examined the potential mechanisms underlying the changes in frequency and power of γ oscillations by recording excitatory currents from an individual PC during network oscillations. They observed that an increase in PC discharges was achieved when PCs received excitatory inputs earlier (~200°) than the expected PC firing. Thereby, PCs were brought above threshold earlier during the γ cycle. Finally, the authors demonstrated that oscillatory frequency could be reduced if the excitatory input arrived at the peak of PC activity. Although the manuscript is very well written and the study well performed, the reviewers formulated some concerns which the authors should address.Essential revisions:1) Although it was examined how excitatory currents are changed as a result of the closed loop situation, it is unclear how inhibitory currents changed. The authors suggest that increased excitation may enable pyramidal cells to fire earlier relative to the periodic inhibition. It is possible, however, that the phase of inhibition also changed relative to the LFP considering the increased contribution of the excitatory currents to the LFP. To fully understand how the γ circuit oscillator functioned during the frequency increase would require describing the timing of inhibition as well relative to the LFP.2) Perhaps the authors could provide an explanation of how the frequency of oscillations could be reduced by their manipulations. Perhaps, by extending the firing of pyramidal cells, interneuron activity could be also prolonged and delayed? Again understating inhibitory dynamics would give a more complete picture about the state of the circuit in generating different frequency γ-band oscillations.3) Is the difference in the shape of the LED command depending on the contribution of the original LFP signal (k1) and its first derivative (k2) . This feature of the feedback signal makes systematic analysis or comparison of phase dependent changes quantitatively difficult, if not impossible.4) Finally, another related aspect which needs clarification is the impact of the relatively slow kinetics of the optogenetic actuator. As illustrated in Figure 5 this could result in a phase difference/delay of over 180 degrees. Arguably, this phase difference is frequency dependent. In view of this property of the feedback optogenetic excitation, how does the analysis of LED-LFP phase relationship presented in Figure 2 and 3 help the reader to understand the impact and the underlying mechanisms? Clearly, the authors are aware of this issue and present Figure 6 which supersedes Figure 3 and puts the initial conclusions into a new perspective. This aspect needs to be clear to the reader from the beginning on. Essential revisions:1) Although it was examined how excitatory currents are changed as a result of the closed loop situation, it is unclear how inhibitory currents changed. The authors suggest that increased excitation may enable pyramidal cells to fire earlier relative to the periodic inhibition. It is possible, however, that the phase of inhibition also changed relative to the LFP considering the increased contribution of the excitatory currents to the LFP. To fully understand how the γ circuit oscillator functioned during the frequency increase would require describing the timing of inhibition as well relative to the LFP.We recorded inhibitory currents in a subset of cells by holding them at the glutamate reversal potential (~0 mV). Under baseline conditions , the phasic inhibitory currents were much larger than the phasic excitatory currents, as expected from the major role of recurrent inhibition in γ and low recurrent excitatory synaptic connectivity in CA1. The fractional change in amplitude of phasic inhibitory currents with γ clamp was, on average, less than 20% of the fractional change in amplitude of excitatory currents. The change in phase was also much smaller than the change in phase of the excitatory currents. This made it difficult to apply the same analysis procedure as for the excitatory currents. That is, subtracting the vector representing the average unclamped inhibitory current from the vector representing the average clamped inhibitory current in some cases resulted in a vector that pointed in a phase direction that was difficult to interpret intuitively. However, a robust outcome was a tendency for changes in the amplitude of the inhibitory current to covary with changes in the power of the oscillation. A simple mechanistic interpretation is that, by modulating excitatory currents, the optogenetic drive alters the timing of firing of principal neurons, which as a downstream consequence affects the recruitment of interneurons, resulting in parallel changes in γ power and inhibitory currents. We have now added a section summarizing the analysis of inhibitory currents to the end of the Results, with representative examples in Figure 7.2) Perhaps the authors could provide an explanation of how the frequency of oscillations could be reduced by their manipulations. Perhaps, by extending the firing of pyramidal cells, interneuron activity could be also prolonged and delayed? Again understating inhibitory dynamics would give a more complete picture about the state of the circuit in generating different frequency γ-band oscillations.As outlined above, changes in inhibitory current amplitudes appear to track changes in oscillatory power, with relatively smaller changes in phase than the changes in excitatory current. In the original version we allowed an inconsistency in the definition of LFP phase between different sections of the paper. We have now streamlined this so that it is clear that 0° refers to the trough of the LFP, which coincides with the maximal firing probability of pyramidal neurons. The conclusion of Figure 6B is now more intuitive: applying an excitatory drive during the downstroke of the LFP (180° to 360°) phaseadvances the firing of pyramidal neurons and therefore results in an increase in frequency. Conversely, applying an excitatory drive during the upstroke (0° to 180°), with a corresponding decrease in excitation during the downstroke, retards pyramidal neuron firing and decreases the oscillatory frequency. This does not mean that interneurons play no role, but by targeting principal cells with the optogenetic drive, changes in their recruitment is mechanistically downstream.3) Is the difference in the shape of the LED command depending on the contribution of the original LFP signal (k1) and its first derivative (k2) . This feature of the feedback signal makes systematic analysis or comparison of phase dependent changes quantitatively difficult, if not impossible.We agree that because the formula results in spectral distortion, the phase difference between the LFP and the LED for some combinations of k1 and k2 was only an approximation. For want of a better method, we calculated it simply by taking the phase at the frequency corresponding to the maximal magnitude of the cross spectrum. The averaged traces in Figure 2C, D actually accentuate the apparent ambiguity in the phase calculation because they give the impression that each cycle of the LFP was highly stereotyped. In fact, the γ oscillation always fluctuates from cycle to cycle in amplitude and phase, and so any error arising from spectral distortion is unlikely to have a major impact on the conclusions. We now acknowledge that the estimated phase difference was only approximate in the text.4) Finally, another related aspect which needs clarification is the impact of the relatively slow kinetics of the optogenetic actuator. As illustrated in Figure 5 this could result in a phase difference/delay of over 180 degrees. Arguably, this phase difference is frequency dependent. In view of this property of the feedback optogenetic excitation, how does the analysis of LED-LFP phase relationship presented in Figure 2 and 3 help the reader to understand the impact and the underlying mechanisms? Clearly, the authors are aware of this issue and present Figure 6 which supersedes Figure 3 and puts the initial conclusions into a new perspective. This aspect needs to be clear to the reader from the beginning on.We hesitated about the order of the narrative. We opted for showing how the behaviour of the oscillation clamp accords with the phase-response curve previously reported in Akam et al., 2012, and then included the spiking data before finishing with the membrane currents, where the underlying mechanisms are resolved. We agree that this seems a long journey, and have therefore added a paragraph to explain the sequence (at the end of the section reconciling clamp behaviour with the PRC).


Rusa marianna) on Guam during depopulation activities as a model for meeting AVMA standards of euthanasia under field conditions . Deer were shot with a .223 caliber rifle from 10–125 m and approached immediately (<20 s) for assessment. A subset of adult deer was further evaluated for physiological responses including cessation of heart rate, respiration, ocular reflexes, and post-mortem spasms. All deer shot in the brain (n = 132) and upper cervical spine  died immediately due to the destruction of the brain or spinal tissue. Shot placements were all within 1.9 cm of the point of aim . The accuracy and immediate insensibility resulting from targeting of C1—C3 demonstrates that this is an alternative target site when animal positioning is not optimal for targeting the brain, or there is a need to preserve brain tissue . While targeting of C4 –C7 vertebrae (n = 6) was accurate and resulted in immediate incapacitation, the failure to produce immediate insensibility does not support the use of this shot placement when upper cervical or brain shot placement is an option. It is reasonable to achieve sufficient accuracy to target the brain or upper cervical vertebrae of deer under field conditions and meet standards of euthanasia while accomplishing management objectives.Population reduction or eradication of domestic or non-domestic species may be required to address their impacts on the environment, other species, or human interests. Firearms are often used to accomplish these practical management objectives, and there is increased concern that the methods used may compromise animal welfare. We document the accuracy and humaneness of gunshot placement to the brain and cervical vertebrae of Philippine deer ( There is a general societal expectation that the taking of animals’ lives should be conducted as humanely as possible. The American Veterinary Medical Association (AVMA) defines euthanasia as a means of ensuring that animals experience a “good death” . The AVMWildlife professionals often use firearms to terminate animals’ lives during population reduction or disease sampling activities. A corollary to this activity is the perspective that wildlife professionals should receive training and achieve levels of proficiency to ensure that firearm use results in a humane death . This peOur primary objective was to determine if gunshot placement to the cranium could be reliably accomplished by trained biologists from ≤125 m and would result in euthanasia as defined by the AVMA. Our secondary objective was to determine if gunshot placement to the cervical spine also would result in euthanasia. We hypothesized that upper cervical spine shot placement would result in euthanasia given the anatomy, particularly vital nerve sources and proximity to the medullary and pontine centers, and that lower cervical spine shot placement may result in lack of unconsciousness and prolonged times to death. We assessed lower cervical shot placement given that it is an often-used method by wildlife professionals  and we wanted to demonstrate the relative humaneness. This research will better define the parameters that result in euthanasia when using firearms. Other shot placements may be necessary to meet management objectives and result in a “humane kill.” Regardless of project goals, killing should be accomplished with the most rapid time to unconscious followed by death.2. According to the 2017 census the island has a human population of 164,229. The Navy Base Guam Naval Munitions Site (NBG NMS) is centrally located on the island and contains some of the highest quality remaining native forest and savanna grasslands on Guam. NBG NMS covers a land area of 5,723 ha and provides habitat for 11 Endangered Species Act (ESA)-listed or -candidate species [Guam is an unincorporated territory of the United States located in the Mariana island chain. The island is the largest in the chain with a land area of 549 kmBoiga irregularis), and the added impact of deforestation caused by non-native ungulates on native species [Data collected for this research were part of a greater initiative with the objective of providing a model for an efficient approach that could be applied to future eradication of non-native ungulates on Guam, particularly the Philippine deer. Philippine deer are native to the Philippine Islands  and were species . There wIntensive population control efforts to reduce overall numbers of non-native ungulates in select areas.Disease surveillance to better understand the risk to humans, domestic animals, and wildlife , 24.Provide recommendations and assistance in planning future non-native ungulate control efforts.http://www.whitebuffaloinc.org). The sample sizes were based on what was practical as a part of field operations, where the client had the objective of assessing optimal methods to depopulate non-native ungulates on NBG NMS. Data were collected on four wildlife biologists who received firearm-specific training for remotely euthanizing animals.We collected data as a part of depopulation efforts conducted by White Buffalo Inc. . Core content included safety, complete understanding of equipment (including equipment maintenance), proper firearm and ammunition selection, and shot placement to kill efficiently and humanely. Other topics included: 1) shooting skill quantification and development, 2) shooting position assessment and training, 3) development of rapid, short-range, multiple target acquisition shooting skills, 4) shooting from elevated locations , 5) night shooting with artificial light, as well as use of thermal and night-vision optics, 6) animal behavior manipulation , and 7) decision-making and situational awareness.Each firearm training course participant was required to pass a post-training field test to be qualified/certified (NWCOA-SISE Level 2) to humanely kill or euthanize animals under field conditions. Field testing requirements were as follows: consistent demonstration of safe firearm handling and backdrop assessment, conservative decision-making while shooting, full awareness of individual and selected equipment limitations, and demonstration of advanced firearm knowledge and skills for use in complex situations. The shooting test required five shots be completed with a centerfire rifle under 35 s (30 s for semi-automatic firearms) at ranges from 10–100 m with <1.9 cm error from point of aim (POA) . Targets were spaced at 10, 25, 50, 75, and 100 m within a 40 m wide shooting zone. Alternatively, the completion of five shots with a rimfire rifle (or air rifle) under 30 s (25 s for a semi-automatic firearm) at ranges from 10–50 m with <1.9 cm error from POA was also acceptable. Targets were spaced at 10, 20, 30, 40, and 50 m within a 40 m wide shooting zone. All shots taken during testing were required to be of high percentage (>98%) based on demonstrated accuracy during individual course evaluations. A final certification (NWCOA-SISE Level 3) was awarded after completion of an apprenticeship under a Level 3 professional. Successful completion of Level 3 training permitted a graduate to operate independently in developed areas. The NWCOA certification requires renewal every two years to help ensure that shooter’s skills are maintained.Deer were approached at night using a vehicle and a spotlight and shot at a distance of 10–125 m, using a suppressed, bolt-action .223 caliber scoped rifle (Leupold VX3 4.5 X 14) with a mounted bipod . The vehhttps://vetmed.iastate.edu/vdpam/about/production-animal-medicine/dairy/dairy-extension/humane-euthanasia/humane-euthanasia/anatomical-landmarks). However, the center of the brain was targeted with a <1.9 cm radius error, as is standard during our wildlife depopulation projects. However, a subset of adult individuals was selected to evaluate the effects of targeting the upper cervical vertebrae  and lower cervical vertebrae  because conventional depopulation methods may target these shot placement locations. Evaluation of deer shot in the lower cervical vertebrae was ceased when it was clear that these individuals were not experiencing immediate insentience, and exhibited extended periods of consciousness until death. Deer shot in the lower cervical vertebrae (C4—C7) that were still alive upon arrival were not terminated with a second shot, so the time it took for the animals to expire could be documented [The POA selected for most deer in this study was the cranium (n = 132) because the goal was the destruction of the brain so that an immediate and humane death  could be ensured. The specific cranial POA chosen varied based on deer’s positioning (see Deer and Pig sections at cumented . These aEach deer in this study (n = 156) was sequentially assigned a unique identification number. A subset of deer were immediately approached (<20 s) after shooting to evaluate several parameters. Evaluation of time to insentience and death were assessed through the following: 1) anatomical impact (site and extent of trauma), 2) POA , and 3) response after being shot (time to recumbency). Physiological parameters that were measured included time until cessation of heart rate, respiration, ocular reflexes , the presence of a glazed eye appearance without tracking, and the cessation of whole body and limb post-mortem spasms . In addiA subset of deer was investigated post-mortem via digital radiography  to evaluate shooter accuracy and terminal ballistic impacts on select tissue. Individually identified deer in each cohort (brain (n = 2), C1 –C3 (n = 14), and C4 –C7 (n = 6)) were sequentially sampled by removal of the head and neck at approximately the C7—T1 junction for assessment and association with field data. These head/neck samples were sealed in individual, leak-proof bags, using methods to minimize biosecurity risks. These measures included personal protective equipment and treatment of exposed areas with disinfectants. These samples were subsequently transported to the Anderson Air Force Base veterinary clinic, and orthogonal views of tissues adjacent to point of impact (POI) were obtained using post-mortem digital radiography . All deeDescriptive statistics were calculated for the time in seconds until cessation of heart rate, respiration, and ocular reflexes, as well as the presence of a glazed eye appearance without tracking, and the cessation of whole body and limb post-mortem spasms. Our data included samples from cranial shots (n = 3), upper cervical spine shots (n = 18) and lower cervical spine shots (n = 6). Data was dropped when NA was listed as the value on an analysis-by-analysis basis.We analyzed our data using SPSS (version 25.0). Depending upon the data distribution, statistical analysis was performed either with Mann-Whitney U test or Student’s two-sample t-test. Shapiro-Wilk tests were used to assess normality. All tests were two-tailed and significance was determined at the 0.05 level. A Mann-Whitney U test was applied to compare the variable of respiration stop time for upper cervical spine shots and lower cervical spine shots. A Student’s two-sample t-test was applied to compare the variable of cessation of eye reflex for upper cervical spine shots and lower cervical spine shots. Heart rate was not compared between treatments as it is not an indicator of immediate insensibility or death  but was All research was conducted under contract with the U.S. Navy (Cooperative Agreement N40192-14-2-8000), and all animal collection procedures were reviewed and approved by University of Georgia’s Institutional Animal Care and Use Committee (A2014 09–021).The weather conditions were similar on all nights of operations; mostly clear skies with intermittent rain events and temperatures from 25-27° C. Deer were not shot when there was precipitation to minimize impacts on equipment and decrease the risk of complications during shooting.All deer shot with the cranium (n = 132) as the POA were killed instantaneously, and no follow-up shots were required (Dataset in Shot placement to C1 –C3 (n = 18) resulted in the immediate unconsciousness and absence of respiration in all animals, as well as profound terminal tissue impacts that are illustrated in Shot placement to C4 –C7 (n = 6) resulted in the immediate absence of respiration in only one animal that was shot at the junction of C3/C4, whereas respiration continued for 55 s to 260 s in the other five animals. Respiration stop times for lower cervical spine shots (mean rank = 17.25) were significantly longer than for upper cervical spine shots (mean rank = 8.5) . No animals were killed instantly. Although all deer were rendered immediately recumbent and incapacitated  with C4—C7 shot placement . There This study describes targeting of the cranium for field euthanasia with gunshot of 132 Philippine deer and successful use of this method at distances of 10 m to 125 m under field conditions. The instantaneous destruction of brain tissue in these deer meets euthanasia criteria established by the AVMA [Targeting of C1—C3 vertebrae also was accurate and resulted in prompt insensibility and death, thereby providing an alternative target site when animal positioning is not optimal for targeting the cranium, or when there is a need to preserve brain tissue. With the increasing prevalence of Chronic Wasting Disease (CWD), and the need to perform detection sampling or depopulation to reduce disease spread, the value of these data becomes more relevant. To validate the presence of CWD, the obex is often tested if the retropharyngeal lymph nodes do not test negative. Gunshot placement to the cranium, with the select caliber and projectile, rarely impacts the retropharyngeal lymph nodes, but often prevents adequate sampling of the obex. Upper cervical shot placement resulted in the loss of spontaneous respiration, although palpebral and corneal reflexes remained.In contrast to deer shot in the lower cervical spine (C4 –C7), deer shot in the C1 –C3 region did not exhibit spontaneous eye tracking, thereby indicating that these animals were insentient during the time period prior to cessation of all vital signs , 26, 27,While targeting of C4—C7 vertebrae was accurate and resulted in immediate incapacitation, the failure to result in immediate deaths does not support the use of this as a humane site for terminating deer lives if upper cervical or brain shot placement is an option. This is especially of concern if an immediate second kill shot is not possible. Shot placement to C4 –C7 resulted in the immediate absence of spontaneous respiration in only one animal, shot at the junction of C3/C4. All deer in this group were aware of our presence (eyes could trace movements) and had erratic breathing and subsequent agonal gasps. There does not appear to be adequate hydrostatic shock when the C4-C7 region is targeted to render an animal unconscious , and theDestruction of ≥30% of brain tissue likely results in the immediate death of animals . TargetiKey to this study was training in applied firearm remote euthanasia. Our shot placement success was greater than those documented when night shooting impala, which achieved 93% successful outcomes , and simAs a consequence of this training, appropriate firearms and ammunition were used in this study to cause a rapid, humane death , 12. We This study documents that it is reasonable for professional wildlife biologists to achieve sufficient accuracy to target the brain or upper cervical vertebrae of deer under field conditions and thereby meet standards of euthanasia while effectively terminating deer lives. We deliberately avoid use of the term “sharpshooter,” as this may be taken to imply that shooters must reach a level of proficiency that most biologists cannot practically achieve. Counter to this misperception, we have documented that biologists can achieve high levels of accuracy with practical levels of training and practice. Although the four biologists that participated in this study had extensive experience (5 to 21 years using the described methods), most training course participants with basic marksmanship skills can achieve levels of proficiency adequate for some field euthanasia scenarios after completion of the two-day course. For example, we have trained seven US law enforcement agencies and one provincial wildlife agency  to successfully euthanize free-ranging deer using this protocol, with many law enforcement participants being certified only in pistol usage prior to the training. Few participants were unable to establish the necessary skills to humanely and efficiently euthanize deer. All could effectively and reliably euthanize single animals, but a few were unable to apply the methods when multiple animals were present in a dynamic setting. This is important because extensive training and experience are not required before biologists are proficient in the use of gunshot as a means of remote euthanasia for wildlife under many field conditions. However, an apprenticeship under advanced level personnel is advised to accelerate further skill development. An equal level of proficiency is required to target the three anatomical sites used in this study . This level of proficiency is relevant for remote field euthanasia of other species, with firearm and bullet selection accommodation for the relative size of the anatomical features targeted and operator awareness of species-specific anatomy, based on our experience euthanizing other ungulates with firearms. We have experienced the same outcomes for cranial and upper cervical shot placement in feral pigs, feral goats, as well as white-tailed, black-tailed, axis and fallow deer. More research on the specifics of caliber, bullet weight, and muzzle velocity is needed for larger species. Of equal importance is that immediate insentience and rapid death meet the expectations of society at large for a humane death, and this can contribute to acceptance of wildlife management plans that require termination of animals’ lives.An additional benefit of targeting the cranium and upper cervical vertebrae, versus the lower cervical spine, is that dispersion of metallic fragments from bullets is limited. This appears to present less risk to humans and animals that consume thoracic musculature from these carcasses, compared to lead deposition patterns of hunter-harvested venison where the thoracic cavity is targeted , 34. WheThe terminal ballistic properties of the select projectiles significantly decreased pass-through safety risks. The lack of an exit wound with upper cervical shot placement also minimized the shedding of blood and tissue, which is especially important when there is a disease concern. Additional data should be collected for a spectrum of calibers  and projectiles to evaluate the relative effectiveness and humaneness of lower energy projectiles, particularly for upper cervical shot placement. It is evident, based on the terminal ballistics and effectiveness of the methods used, that there is no value in increasing the size or energy of the projectiles for species of similar stature. This is because larger or more robust projectiles only increase the risk of bullet pass-through and thereby decrease the safety of depopulation programs.This study’s results should not be interpreted within a limited scope for depopulation of cervids. Training and gunshot accuracy is relevant to all ungulates on the basis of the authors’ experience. Consequently, these methods also are relevant for training emergency responders for disease control depopulation  and free-ranging animals affected by severe weather conditions and other natural disasters , as well as for other purposes. Collection of additional data on other species and under other conditions is ongoing.Our objective was to document that gunshot to the brain of free-ranging deer can be consistently achieved and results in immediate insentience as a means of remote euthanasia. We also assessed the accuracy and outcomes of targeting cervical vertebrae as alternative shot placement options under field conditions that would result in euthanasia without compromising program objectives . The target site that we evaluated provided useful animal welfare data for management programs where immediate incapacitation is the primary objective for public safety and program efficiency. We documented the ramifications of various shot placements, based on anatomical expectations that would result in incapacitation. This will guide professionals using firearms to kill animals to ensure that they are aware of the relative humaneness of shot placement decisions.Rusa marianna) in Guam. The training that biologists received and methods employed were practical and reasonably attainable for achieving high levels of gunshot placement accuracy under field conditions. We documented that shots were successfully targeted within a 1.9 cm diameter, that all deer receiving cranium gunshot placement were instantly insentient, and that the methods we used met AVMA standards of euthanasia despite being conducted under field conditions [This study documents the success of wildlife biologists trained to accurately shoot deer under field conditions at distances of 10–125 m, as part of a contracted population reduction program for Philippine deer (S1 Dataset(PDF)Click here for additional data file.


At the edge of the hypertrophic zone, chondrocytes re-enter the cell cycle and express leptin receptor (lepr), suggesting conversion into progenitors. Further, mutation of matrix metalloproteinase 9 (mmp9) results in delayed growth plate remodeling and fewer marrow adipocytes. Our data support Mmp9-dependent growth plate remodeling and conversion of chondrocytes into osteoblasts and marrow adipocytes as conserved features of bony vertebrates.Much of the vertebrate skeleton develops from cartilage templates that are progressively remodeled into bone. Lineage tracing studies in mouse suggest that chondrocytes within these templates persist and become osteoblasts, yet the underlying mechanisms of this process and whether chondrocytes can generate other derivatives remain unclear. We find that zebrafish cartilages undergo extensive remodeling and vascularization during juvenile stages to generate fat-filled bones. Growth plate chondrocytes marked by  Our adult bones are made of a fatty tissue, called marrow, wrapped inside a hard outer layer produced by bone cells. They may appear stiff and unyielding, but our bones are actually dynamic structures. Early in life, most bones start as small ‘templates’ made of another, flexible tissue called cartilage. As the templates grow into adult bones, the cartilage is gradually replaced by bone and fat, but this process is still poorly understood. For example, it is not clear whether cartilage cells simply die and make way for new cells, or instead if they turn into bone and fat cells. To investigate this question, Giovannone, Paul et al. set out to follow the fate of early cartilage cells in zebrafish, and to compare this with what happens in mammals. Zebrafish were chosen because their skeleton and ours develop in similar ways; yet, these animals are much easier to study, in particular because their embryos are transparent.Young cartilage cells were ‘tagged’ with a long-lasting fluorescent protein in genetically engineered zebrafish embryos, and then followed over time. As the embryos started to form bones, the cartilage cells gave rise to bone cells, fat cells, and also potentially adult stem cells within the marrow, which can become other types of cells. This process required a protein called Mmp9, which also helps shape bone development in other organisms, including humans.Defects in how early cartilage templates morph into bone and fat may contribute to dwarfism and other severe conditions. Fully grasping the molecular mechanisms that preside over this complex transition may one day help design drugs to treat skeletal disorders. Vertebrate bones develop via two largely distinct processes. Intramembranous  bone, which makes up a large portion of the skull, arises through the direct differentiation of mesenchymal precursors into osteoblasts and then osteocytes. In contrast, endochondral bone, which comprises the majority of the axial and limb skeletons, arises through the progressive remodeling of an embryonic cartilage template. On the outside of developing endochondral bone, perichondral cells mature into periosteal progenitors that contribute to the bone collar. The cartilage templates of endochondral bone are organized into distinct zones of chondrocytes: resting, proliferative, pre-hypertrophic, and hypertrophic. In mammals, chondrocytes at the edge of the developing hypertrophic zone largely disappear as the cartilage matrix is degraded, a process concurrent with the invasion of blood vessels, hematopoietic cells, and progenitors for osteoblasts and marrow adipocytes . This grCol10a1-Cre- and Aggrecan-Cre-based transgenes in mice, has revealed that such transdifferentiation may also occur in vivo, with chondrocytes making a major contribution to osteoblasts within trabecular bone and potentially also the bone collar  bone of the lower face as a model. This long bone, which is derived from cranial neural crest cells, exhibits properties in common with the long bones of mammalian limbs, including two prominent growth plates at either end and a marrow cavity . Here, wer birth . In zebrmodeling , we findIn order to characterize the progressive remodeling of an endochondral bone in zebrafish, we performed pentachrome staining on sections of the Ch bone from juvenile through adult stages . The Ch col2a1a:mCherry-NTR transgene and fli1a:GFP , the Ch is heavily supplied with both kdrl:GFP+blood vessels and lyve1:DsRed+lymphatic vessels, which abut each side of the growth plate . The zebrafish sox10 promoter drives expression in early cranial neural crest cells from 10 to 16 hpf, followed by a second wave of expression in all chondrocytes from two dpf onwards (sox10/B > R fish by 4-OHT treatment at 14 dpf and raised these to adulthood (27 mm SL) for analysis, with inclusion of an ocn:GFP transgene allowing us to label osteoblasts   . When cr:CreERT2 . Analysi surface . This is surface . However surface .sox10:CreERT2-mediated conversion at 14 dpf also labels cells outside the cartilage, which could also contribute to osteoblasts and adipocytes, we next examined animals with lower conversion efficiency to follow discrete growth plate clones. When analyzed at 30 mm SL, growth plate clones could be quite large, consistent with clonal selection as described in the zebrafish heart and skeletal muscle  to various color combinations of CFP, YFP, and RFP at 23 mm SL. Analysis of uniquely colored clones in the growth plate revealed those that contained both chondrocytes and adjacent adipocytes in the marrow , maximal intensity projections through Ch revealed extensive labeling of growth plate chondrocytes, as well as cells throughout the bone and marrow cavity  can result in strong reporter expression. In zebrafish, col2a1a is expressed at high levels in the proliferative zone of the Ch growth plate and then downregulated in the hypertrophic zone  and/or through partial dedifferentiation into a proliferative progenitor. To test these possibilities, we first used incorporation of bromodeoxyuridine (BrdU) to detect proliferative cells in the Ch during remodeling stages . At the LepR expression marks a heterogeneous population of cells in endochondral bone, including a putative postnatal skeletal stem cell population   . These rMmp9 has been reported to function in hematopoietic lineage cells for growth plate remodeling, as bone marrow transplants can rescue the delay in growth plate remodeling seen in Mmp9 mutants , we observe expression of mmp9 at the edge of the hypertrophic zone, with this restricted expression in late-stage hypertrophic chondrocytes continuing through 17 mm SL stages , as well as a trend toward rescue of adipocyte number (p = 0.06)  . As a commp9 mutants results in a paucity of marrow adipocytes in adults. Lastly, we show that hypertrophic chondrocytes re-enter the cell cycle and contribute to lepr+ mesenchymal cells, raising the possibility that partial dedifferentiation into proliferative progenitors underlies chondrocyte fate transitions inside endochondral bones.Despite anatomical differences between zebrafish and mammalian bones, we find that growth plate remodeling is remarkably well conserved and thus likely ancestral to bony vertebrates. The zebrafish Ch undergoes a transient breakdown of cortical bone near the growth plates, which coincides with extensive vascularization and an Mmp9-dependent replacement of hypertrophic chondrocytes with fat and bone. Using multiple methods of lineage tracing, including a Cre-independent technique, we show that late-stage hypertrophic chondrocytes generate not only osteoblasts but also marrow adipocytes in zebrafish. Further support for the ability of chondrocytes to generate adipocytes is that delayed growth plate remodeling in sox10:CreER outside of chondrocytes, made it difficult to precisely quantify what proportion of osteoblasts and marrow adipocytes derive from chondrocytes, there are likely other sources for these cells, in particular the periosteum which houses several types of skeletal stem cells . Indeed, the detection of osteoblasts with strong col2a1a:Histone2A-mCherry signal suggests that in some cases chondrocytes can form osteoblasts with little to no cell division, as otherwise the Histone2A-mCherry signal would have been diluted out with successive cell divisions. On the other hand, osteoblasts farther from the growth plate tended to have weaker Histone2A-mCherry signal, suggesting proliferation of a progenitor intermediate, and we directly observed late-stage hypertrophic chondrocytes undergoing cell division. Whereas it has been suggested in mouse that only chondrocytes near the periosteal surface, that is ‘borderline chondrocytes’, may be capable of lineage plasticity  that displays more restricted expression than the short isoform , potentially owing to low sample size (n = 8), the mosaic contribution of wild-type cells to the growth plate, and/or roles of cells outside the growth plate to marrow adipocyte generation. In mice, loss of Mmp13 in chondrocytes does result in a growth plate remodeling delay, with global Mmp13 deletion enhancing the remodeling defects of Mmp9 mutants  and zf384Tg(Mmu.Sox10-Mmu.Fos:Cre) (b1212Tg(sp7:EGFP)  (el483Tg(col2a1aBAC:GFP) and el559Tg(col2a1aBAC:mCherry-NTR) (sd27Tg(bactin2:loxP-BFP-loxP-DsRed) (y1Tg(fli1a:eGFP) (s843kdrl:eGFP) (nz101Tg(−5.2lyve1b:DsRed) (a131Tg(ubb:LOX2272-LOXP-RFP-LOX2272-CFP-LOXP-YFP) (sox10:CreERT2 transgene was generated with Gateway Cloning (Invitrogen) and the Tol2 kit . H2A.F/Z-mCherry and P2A-GFPCAAX were combined with an NcoI/EcoRI fragment of pDestTol2-col2a1aR2-E1B-eGFPpA using Gibson Assembly. All transgenes were injected at 30 ng/μL along with 50 ng/μL Tol2 mRNA into one-cell-stage embryos, with these animals raised and outcrossed to identify stable germline founders. CreERT2 transgenes were injected into sd27Tg(bactin2:loxP-BFP-loxP-DsRed) embryos, followed by overnight treatment with 10 μM (Z)−4-Hydroxytamoxifen (4-OHT) (Sigma-Aldrich H7904) at two dpf and screening for DsRed+ chondrocytes at six dpf using a Leica fluorescent stereomicroscope. Embryos with fluorescent cartilages were raised to adulthood and outcrossed to identify founders. We used el777Tg(sox10:CreERT2), el712Tg(col2a1a-R2-E1b:CreERT2), and el695Tg(col2a1a-R2-E1b:H2A.F/Z-mCherry-P2A-GFPCAAX) lines for our experiments. Two additional alleles of Tg(col2a1a-R2-E1b:CreERT2) and one additional allele of Tg(col2a1a-R2-E1b:H2A.F/Z-mCherry-P2A-GFPCAAX) gave similar cartilage-specific expression. To generate el734mmp9 we used CRISPR/Cas9 mutagenesis to target the second exon. gRNA targeting the sequence 5’-TTGATGCCATGAAGCAGCCC-3’ was injected at 25 ng/μl with 50 ng/μl Cas9 RNA into one-cell-stage embryos as described , followed by a premature stop codon that is predicted to completely abolish the catalytic metalloproteinase domain.All procedures were approved by the University of Southern California Institutional Animal Care and Use Committee. Published re)zf384 , Tg(sp7:FP)b1212 , Tg(Ola.P)hu4008 , Tg(col2TR)el559 , Tg(bactRed)sd27 , Tg(fli1:eGFP)y1 , Tg(kdrlGFP)s843 , Tg(−5.2ed)nz101 , and ZebYFP)a131 . The soxTol2 kit  by combiTol2 kit , pME-Cresequence . For colB-eGFPpA  using Giescribed . The mmpLive bone staining of dissected ceratohyal bones was performed by treating with 50 µg/ml Alizarin Red , Calcein Green , or Calcein Blue, AM  for 5 min and repeatedly rinsing in embryo medium as described . We perfmmp9 riboprobe was generated by PCR amplification of zebrafish genomic DNA with primers 5’-GTCTCCAATACTAAAGCTCTGAAGAAG-3’ and 5-‘TAGGATGTCGAAGGTCTATAGAGAATG-3’ and cloning into pCR-BluntII-TOPO (Life Technologies). RNA probe was synthesized with T7 polymerase (Roche) after linearizing the plasmid with BamHI restriction. RNAscope probes for leptin receptor (lepr) and runx2b were synthesized by Advanced Cell Diagnostics in Channel 1 and Channel 2, respectively, and for mouse Lepr in channel 1. Paraformaldehyde-fixed paraffin-embedded sections were deparaffinized, and the RNAscope fluorescent multiplex v2 assay combined with immunofluorescence was performed according to manufacturer’s protocols and with the ACD HybEZ Hybridization oven.For cell proliferations assays, fish were immersed in system water containing 4.5 mg/ml BrdU  for 1 hr, followed by two washes in system water, fixation in 4% paraformaldehyde, and paraffin embedding. Immunohistochemistry was performed as described except that we blocked with 2% normal goat serum . After cutting thin sections, we performed antigen retrieval by treating slides with citrate buffer (pH 6.0) in a steamer set  for 35 min. Primary antibodies include rat anti-BrdU  and rabbit anti-mCherry . We used AlexaFluor secondary antibodies and Hoechst to visualize nuclei. Colorimetric in situ hybridization was performed as described . The mmpbactin2:loxP-tagBFP-loxP-DsRed embryos was transplanted into the neural crest precursor domain of -/-mmp9 hosts as described  from the tip of the snout to the edge of the hypuralia. Adipocyte counts and area/width measurements of growth plates were calculated using Fiji. The proliferative zone of the growth plate was defined as the central region of compact chondrocytes. All measurements were performed blinded to genotype. Statistical significance was determined by a student’s t-test for pair-wise comparisons or Tukey-Kramer HSD tests for multiple comparisons, using GraphPad’s Prism software. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Didier Stainier as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: David Maridas (Reviewer #2).Thank you for submitting your article "Programmed Dedifferentiation of Hypertrophic Chondrocytes Generates Osteoblasts and Adipocytes in Zebrafish Bones" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.LepR+ cells is required since the authors conclusions differ from the kinetics in mice, raising an important distinction if true between fish and mammals. The comparisons between species however, are further limited by the difference in the LepR isoform used in the Cre lines between those of Morrison and in this work. Second, several components of Figure 3 are not clear and raise questions about labeling and timing of 4OHT- this aspect will require more experiments. Third, the results from the MMP9 mutation are not directly related to the major tenet of the manuscript and the rescue experiments are based on small numbers of fish. Finally, it is conceptually difficult to appreciate the authors conclusions that the marrow adipocytes represent a fat reserve and that hematopoiesis follows later- there are no data to support that assumption. Are the authors suggesting the adipocyte depot represents a place holder for subsequent hematopoiesis?Reviewing Editor: All agree the work is novel and represents potentially important differences with the more frequently studied mammalian systems. As such there is support for the manuscript. But there are several limitations that need to be addressed in a revision. First a clearer characterization of As such these four points plus other comments from the reviewers will require more experiments and a comprehensive revision.Reviewer #1:This elegant and well written manuscript describes how chondrocytes in the zebrafish CH cartilage template gives rise to osteoblasts and adipocytes during CH bone maturation. The studies show that the CH cartilage template undergoes remodeling and vascularization during juvenile stages to generate fat-filled bones in the adult. Growth plate chondrocytes re-enter the cell cycle, and express the progenitor cell marker leptin receptor, suggesting dedifferentiation into progenitor cells, and then re-differentiation into osteoblasts, adipocytes and mesenchymal cells within the adult CH.This study answers the important question as to whether zebrafish bone arises through invasion of the vasculature and conversion of growth plate cartilage to bone, as occurs in mammals. And also whether hypertrophic chondrocytes directly transform into osteoblast, or do so through a stem cell intermediate. This study identifies growth plate chondrocytes as a source of osteocytes and adipocytes in zebrafish bones, and suggest that dedifferentiation into proliferative progenitors as the likely mechanism.The authors describe the bidirectional growth of the zebrafish CH growth plates , and use beautiful lineage labeling studies to show chondrocyte clonal cell contributions to mesenchymal cells and adipocytes – and possibly osteoblasts – after growth plate remodeling.lepr was used to monitor hypertrophic chondrocyte differentiation and de-differentiation into a progenitor cell-like state.Multiple approaches are used to demonstrate that hypertrophic chondrocytes and their osteoblast derivatives undergo cell division, indicating a dedifferentiation of hypertrophic chondrocytes. Conversion of hypertrophic chondrocytes to osteoblasts/adipocytes via transdifferentiation or through partial de-differentiation into a proliferative progenitor was examined using BrdU labeling. The expression of the progenitor cell marker Mmp9/13 in this process.The authors conclude that growth plate remodeling is a conserved feature of bony vertebrates, and that chondrocyte dedifferentiation is a mechanism to generating osteoblasts and marrow adipocytes in the zebrafish. They also describe roles for eLife.I recommend that this manuscript be accepted for publication in Strengths:1) Beautiful imaging, well described and careful studies.2) The finding that late-stage hypertrophic chondrocytes give rise to osteoblasts and marrow adipocytes in the zebrafish CH.3) Chondrocytes also give rise to both endosteal and periosteal surfaces fo the Ch bone, similar to mouse.mmp9 is required, in the neural crest cell lineage and perhaps in chondrocytes themselves, for Ch growth plate width, remodeling and adipocyte number, using both mmp9 mutant and rescue by WT cell transplantation into mmp9 mutant zebrafish.4) Demonstration that 5) The finding that growth plate remodeling is well conserved between mammals and fishes.6) Use of three different methods of lineage tracing, including a Cre independent technique, to show that late-stage hypertrophic chondrocytes generate not only osteoblasts but also marrow adipocytes in zebrafish.7) The interesting hypothesis that evolutionarily, fish bone marrow may represent the ancestral condition of being primarily a fat reservoir, with hematopoiesis moving into the bone later during tetrapod evolution.mmp9 may have additional non-canonical function in the nucleus for histone H3 tail cleavage in hypertrophic chondrocytes, as has been demonstrated for osteoclasts.8) The intriguing hypothesis that lepr expression patterns is discussed, and proposed that this may be due to the more restricted expression of the long LepR isoform (mouse) as compared to the short isoform. Direct analyses of the two isoforms in zebrafish is proposed.9) Discrepancies with the mouse Reviewer #2:This manuscript elegantly demonstrates the significant contribution of hypertrophic chondrocytes to bone marrow fat in zebrafish. The authors also provide solid data to back their conclusions and suggest a novel mechanism of de-differentiation.However, I have two comments about the context and the data presented in this article:1) Although they mentioned the possibility that chondrocytes differentiate into adipocytes in mammals in the discussion, the authors missed on placing their findings with the current literature in mammals, notably mouse studies. Murine chondrocytes cultures can differentiate in adipocytes in vitro. Also, Ono et al., 2014, have previously showed that Col2CRE-ER dtomato mice do not show adipocyte labelling at 1 week, 1month or even 1 year after tamoxifen induction. It would be interesting for the authors to mention whether this specific to fish or not.2) Would it be possible for the authors to quantify the contribution of chondrocytes to the total number of adipocytes? In other words, what is the ratio of labelled vs non-labelled adipocytes in their adult animals?mmp9 mutants because of the low n in that experiment (i.e. n = 2)? If so, the authors should mention the limitation of a low n.3) Is it possible that WT donor cells coming from WT did not rescue the number of adipocytes in Reviewer #3:In this manuscript, Giovannone et al. examine the results of neural crest and cartilage lineage tracing in a zebrafish model, that chondrocytes can contribute to mesenchyme in the endosteal compartment. Overall, the observations made here mostly mirror previously published studies in mouse systems. While there are novel insights to be gained from further lineage tracing studies in a zebrafish system to understand if these findings are conserved, the existence of extensive prior literature utilizing this approach in mice generally limits the additional novel insights to be gained unless the findings of this system are pursued in substantial depth, which is largely not the case in the present manuscript.Col2a1 system performed here is very restricted compared with use of similar reagents in mice. This is of interest for suggesting that the presence of chondrocyte-like gene expression signatures in bona fide MSCs is restricted to mammalian systems and therefore a recent evolutionary development. However, this is not explored in sufficient detail to flesh out this possibility and draw clear conclusions on this point. Similarly potentially interesting observations are made regarding the origin of LEPR+ cells, however these are not explored in sufficient detail to provide clear answers as to the origin of marrow resident LEPR cells. Additional specific comments are described below:A few potentially significant novel observations are made but not followed up upon. For example, the labeling with the Major points:Col2a1 system addresses some of these weaknesses of the Sox10 system.1) It is unclear if the timing of 4-OHT administration truly restricts Sox10-cre ERT driven recombination to cartilage and not to other populations of neural crest-derived progenitors of the Ch bone. Indeed, the labeling of the periosteum observed suggests that the latter possibility is more likely. Given that Sox10 may label neural crest-derived mesenchymal progenitors beyond just chondrocytes, the use of Sox10-based lineage tracking is problematic for experiments designed to inform on chondrocyte conversion into osteoblast or adipocyte-lineage cells. The comparison to the broader deletion mediated by the human Sox10-cre is helpful, but does not exclude labeling of other non-cartilage MSC populations. It is appreciated that the col2a1-mediated labeling is restricted in zebrafish relative to observations made in mice is interesting for suggesting that the presence of a chondrocyte-like gene expression program in MSC populations is restricted to mammals. However, further experiments to directly assess this would be needed to flesh out this possibility.2) The images shown in Figure 3E are not convincing that they represent solely labeling of 3 independent clones as the regions of the distinct clones appear to partially overlap in the image available, undercutting the claim of clonal labeling. Titration studies of 4-OHT are needed to establish the dose ranges where clonal labeling is observed. Moreover even with full validation of a dose titration series, it is impossible to be certain that labeling represents true single clone derivatives unless either serial imaging is performed or a multiclone reporter similar to the confetti mouse is used. The suggestion that Col2a1-cre in zebrafish and observing the actual phenotypic contribution of post-chondrocyte osteoblasts to skeletal mineralization.3) A major limitation of this study is that it only tracks cells but does not assess their functional contribution to mineralizaiton, for instance by deleting a gene required for mineralization using LEPR+ cells is primarily of interest for the suggestion that cartilage is the source of the LEPR+ cell populations described in mice. However, these part of the manuscript has limitations that prevent it from providing substantial insights into the question of the source of the LEPR+ cells as they are described in the murine system.4) The investigation of cartilage cells as a source of LEPR-cre marked cells is that LEPR generally excludes cells in the growth plate. Thus, the significance of LEPR labeling in cartilage in fish for mammalian physiology is unclear. Indeed, the labeling in cartilage appears quite weak relative to the labeling in cells adjacent to bone and it is unclear what degree of signal in this in situ hybridization based system would correspond to the cells marked by LEPR-cre in mice. There is also no characterization of the LEPR+ cells offered, so it is unclear to what degree these cells observed in zebrafish correspond functionally to murine LEPR+ cells. Additionally, a defining feature of murine LEPR osteogenic cells is that they do not emerge until adulthood. What are the kinetics of LEPR+ cells emerging during the lifetime of the fish?Aside from these concerns, one of the notable features of murine MMP9 is not strongly related to the rest of the manuscript. No direct evidence is offered to show that any of the phenotypes seen with the MMP9 loss of function relate to transdifferentiation of cartilage cells.5) The work on 6) In Figure 6, how can it be excluded that the Brdu labeling observed is due to labeling of proliferating chondrocytes that have progressed to the hypertrophic stage by the time of visualization? Reviewing Editor: All agree the work is novel and represents potentially important differences with the more frequently studied mammalian systems. As such there is support for the manuscript. But there are several limitations that need to be addressed in a revision.First a clearer characterization of LepR+ cells is required since the authors conclusions differ from the kinetics in mice, raising an important distinction if true between fish and mammals.lepr at 4 additional stages in zebrafish, as well as in situ analysis of Lepr in the postnatal mouse femur. These experiments show that lepr+ cells in the fish marrow emerge only after remodeling of the growth plate. We also find similar expression of fish lepr and mouse Lepr in chondrocytes at all stages, with expression in both species most prominent in the proliferative zone of the growth plate. We reference previous papers showing similar chondrocyte expression of Lepr in mouse, rat, and human, and that the marrow-specific expression of mouse Lepr-Cre is likely due to this transgene targeting only the long isoform of Lepr. In response to reviewer 3, we have modified the Discussion to stress that our results show that lepr+ marrow cells are derived from chondrocytes, and that further experiments are needed to determine if these are equivalent to the Lepr-Cre+ marrow stem cells reported in mouse.In a new Figure 6—figure supplement 1, we have performed in situ analysis of Second, several components of Figure 3 are not clear and raise questions about labeling and timing of 4OHT- this aspect will require more experiments.sox10:CreER was problematic given its expression in additional non-cartilage cells. In a new Figure 3F, we have now performed more precise clonal labeling using zebrabow fish as requested, which reveals common color clones of chondrocytes and adipocytes, and chondrocytes and putative osteocytes. This strengthens our argument that it is sox10:CreER-labeled chondrocytes that give rise to adipocytes and osteocytes.See response to reviewer 3 for more details.We agree with reviewer 3 that our initial analysis using Third, the results from the MMP9 mutation are not directly related to the major tenet of the manuscript and the rescue experiments are based on small numbers of fish.mmp9 mutant analysis are not directly related to the manuscript. The mmp9 mutant analysis is used to make two major points. First, it was previously unclear whether fish growth plates undergo the same type of remodeling seen in mammalian bones. A common delay in growth plate remodeling in fish mmp9 and mouse Mmp9 mutants therefore supports genetic conservation of remodeling in both species. Second, we show that zebrafish mmp9 functions in chondrocytes themselves for remodeling. By increasing our n number for rescue experiments (from n=2 to n=8 in the revision) we now see that restoring mmp9 in chondrocytes rescuesadipocyte generation . Thus, analysis of mmp9 mutants provides important genetic evidence supporting our major tenet that remodeling of chondrocytes generates adipocytes.We disagree that the results from the Finally, it is conceptually difficult to appreciate the authors conclusions that the marrow adipocytes represent a fat reserve and that hematopoiesis follows later- there are no data to support that assumption. Are the authors suggesting the adipocyte depot represents a place holder for subsequent hematopoiesis?We acknowledge that this evolutionary statement was highly speculative and have therefore removed it from the Discussion.As such these four points plus other comments from the reviewers will require more experiments and a comprehensive revision.Reviewer #2:This manuscript elegantly demonstrates the significant contribution of hypertrophic chondrocytes to bone marrow fat in zebrafish. The authors also provide solid data to back their conclusions and suggest a novel mechanism of de-differentiation.However, I have two comments about the context and the data presented in this article:1) Although they mentioned the possibility that chondrocytes differentiate into adipocytes in mammals in the discussion, the authors missed on placing their findings with the current literature in mammals, notably mouse studies. Murine chondrocytes cultures can differentiate in adipocytes in vitro. Also, Ono et al., 2014, have previously showed that Col2CRE-ER dtomato mice do not show adipocyte labelling at 1 week, 1month or even 1 year after tamoxifen induction. It would be interesting for the authors to mention whether this specific to fish or not.creER-P3 cells continued to yield chondrocytes, osteoblasts and stromal cells in the metaphysis, and also became adipocytes in the metaphyseal bone marrow ”. We now include a discussion of the current mouse literature.We thank the reviewer for alerting us to these previous studies. A re-examination of  revealed that they did in fact observe adipocyte labeling post-chase –“During an extended chase period over a year after the pulse, Col2Col2a1:CreER cells converted at postnatal day 3 in mouse were found to give rise to adipocytes in the metaphyseal bone marrow, although a caveat is that Col2a1:CreER marks both chondrocytes and progenitors at early postnatal stages .”“Indeed, older studies have demonstrated the ability of murine chondrocytes to differentiate into adipocytes in vitro . Further, 2) Would it be possible for the authors to quantify the contribution of chondrocytes to the total number of adipocytes? In other words, what is the ratio of labelled vs non-labelled adipocytes in their adult animals?We considered quantification but concluded that it would not be skewed due to several factors. First, conversion efficiency is variable in our CreER lines. Second, perhaps due to gene silencing or the small amount of cytoplasm in mature adipocytes, we cannot always definitively detect the reporter allele (converted or unconverted) in all adipocytes. Thus, while we could certainly attempt to quantify the proportion of lineage cells, any quantification would be an under-representation of chondrocyte contribution to adipocytes.3) Is it possible that WT donor cells coming from WT did not rescue the number of adipocytes in mmp9 mutants because of the low n in that experiment (i.e. n = 2)? If so, the authors should mention the limitation of a low n.We have now performed additional transplantation experiments and have increased our n from 2 to 8, which reveal a strong trend toward rescue of adipocyte numbers .n = 8), the mosaic contribution of wild-type cells to the growth plate, and/or roles of cells outside the growth plate to marrow adipocyte generation.”“We also observed rescue of marrow adipocyte number by wild-type chondrocytes, though this had moderate statistical significance (p = 0.06), potentially owing to low sample size . This avoids the weak expression of zebrafish col2a1a in perichondrium/periosteum, which we now reference. In the future, a zebrafish col2a1a-CreER knock-in line that reports full col2a1a expression (including in the perichondrium/periosteum) would be needed to test whether col2a1a+ MSCs similar to those reported by  also exist in zebrafish.We thank the reviewer for this comment and agree that we had not previously explained key differences in the way the zebrafish Col2a1:CreERT2-mediated conversion at embryonic and early postnatal stages broadly labels not only chondrocytes but also osteochondroprogenitors, such as those in the perichondrium and periosteum . […]Treatment of col2a1a/B>R fish with a single dose of 4-OHT at 5 dpf resulted in extensive labeling of col2a1a-BAC:GFP+chondrocytes at 12 dpf, but no labeling of the perichondrium, periosteum, and osteoblasts as marked by the sp7:GFP transgene .”“In mice, col2a1a-BAC:GFP but not the osteoblast and perichondrium/periosteum marker sp7:GFP. These additional controls further validate the use of this CreER line to follow the long-term fates of fish chondrocytes in a specific manner.In a new Figure 4A,B, we present additional controls (including high magnification views of the perichondrium) showing co-localization of col2a1a-CreER-converted cells with the chondrocyte marker Major points:1) It is unclear if the timing of 4-OHT administration truly restricts Sox10-cre ERT driven recombination to cartilage and not to other populations of neural crest-derived progenitors of the Ch bone. Indeed, the labeling of the periosteum observed suggests that the latter possibility is more likely. Given that Sox10 may label neural crest-derived mesenchymal progenitors beyond just chondrocytes, the use of Sox10-based lineage tracking is problematic for experiments designed to inform on chondrocyte conversion into osteoblast or adipocyte-lineage cells. The comparison to the broader deletion mediated by the human Sox10-cre is helpful, but does not exclude labeling of other non-cartilage MSC populations. It is appreciated that the Col2a1 system addresses some of these weaknesses of the Sox10 system.2) The images shown in Figure 3E are not convincing that they represent solely labeling of 3 independent clones as the regions of the distinct clones appear to partially overlap in the image available, undercutting the claim of clonal labeling. Titration studies of 4-OHT are needed to establish the dose ranges where clonal labeling is observed. Moreover even with full validation of a dose titration series, it is impossible to be certain that labeling represents true single clone derivatives unless either serial imaging is performed or a multiclone reporter similar to the confetti mouse is used. The suggestion that col2a1-mediated labeling is restricted in zebrafish relative to observations made in mice is interesting for suggesting that the presence of a chondrocyte-like gene expression program in MSC populations is restricted to mammals. However, further experiments to directly assess this would be needed to flesh out this possibility.sox10:CreER given its additional activity outside chondrocytes (e.g. in the perichondrium). In our hands, conversion with 4OH-T at 14 dpf gave the most robust conversion in cartilage, and we have yet to find a time of 4OH-T treatment that only activates in cartilage. We also acknowledgethe limitations of our original clonal analysis given the overlapping nature of the clones. We have therefore performed new experiments using the multicolor zebrabow reporter, which operates similarly to the confetti mouse. New data in Figure 3F now more clearly support clonal contribution of chondrocytes to adipocytes and putative osteocytes.We agree with the reviewer about the limitations of using sox10:CreERT2; ubb:Zebrabow fish in which 4OH-T treatment at 14 dpf resulted in conversion of RFP (red) to various color combinations of CFP, YFP, and RFP at 23 mm SL. Analysis of uniquely colored clones in the growth plate revealed those that contained both chondrocytes and adjacent adipocytes in the marrow, as well as those that contained chondrocytes and cells embedded in cortical bone, consistent with an osteoblast/osteocyte identity .”“To more definitively follow growth plate clones, we also examined 3) A major limitation of this study is that it only tracks cells but does not assess their functional contribution to mineralizaiton, for instance by deleting a gene required for mineralization using Col2a1-cre in zebrafish and observing the actual phenotypic contribution of post-chondrocyte osteoblasts to skeletal mineralization.We agree that this would be an informative experiment in the future. Unfortunately at present, precise integration of loxP sites into the zebrafish genome is technically very challenging. To our knowledge, there is only one report to date claiming to have accomplished this . Even if possible, it would be a lengthy process constructing these fish, and as such we feel attempting this would be well beyond the scope of the current study.4) The investigation of cartilage cells as a source of LEPR+ cells is primarily of interest for the suggestion that cartilage is the source of the LEPR+ cell populations described in mice. However, these part of the manuscript has limitations that prevent it from providing substantial insights into the question of the source of the LEPR+ cells as they are described in the murine system.Aside from these concerns, one of the notable features of murine LEPR-cre marked cells is that LEPR generally excludes cells in the growth plate. Thus, the significance of LEPR labeling in cartilage in fish for mammalian physiology is unclear. Indeed, the labeling in cartilage appears quite weak relative to the labeling in cells adjacent to bone and it is unclear what degree of signal in this in situ hybridization based system would correspond to the cells marked by LEPR-cre in mice. There is also no characterization of the LEPR+ cells offered, so it is unclear to what degree these cells observed in zebrafish correspond functionally to murine LEPR+ cells. Additionally, a defining feature of murine LEPR osteogenic cells is that they do not emerge until adulthood. What are the kinetics of LEPR+ cells emerging during the lifetime of the fish?lepr in situs at 4 additional stages in zebrafish, as well as Lepr in situs in mouse. These new data show conserved expression of fish/mouse Lepr in growth plate chondrocytes, with strongest expression in the proliferative zone. They also reveal emergence of lepr+ marrow cells in zebrafish only after initiation of growth plate remodeling . We discuss that this likely reflects the Lepr-Cre line only reporting expression of the long-isoform version of Lepr.In a new Figure 6—figure supplement 1, we perform lepr expression in both marrow cells and growth plate chondrocytes in zebrafish. However, the more specific labeling of marrow cells by the LepR-Cre in mouse likely reflects the Cre insertion being in the long LepR isoform (containing exon 18b) that displays more restricted expression than the short isoform . Indeed, LepR mRNA and protein has also been reported in chondrocytes of mouse , rat, and human , a finding we confirmed in the postnatal mouse femur, including the same higher expression in immature versus hypertrophic chondrocytes that we observe in zebrafish .”“One caveat is that we detect endogenous LEPR does not in and of itself specify a skeletal stem cell as mentioned in the text. Rather it labels a heterogeneous population of cells  that may include a stem cell subset, though this has yet to be directly addressed experimentally.We have revised the text to provide a more conservative treatment of LEPR as suggested.LepR expression marks a heterogeneous population of cells in endochondral bone, including a putative postnatal skeletal stem cell population … These results are consistent with lepr+ cells in the bone marrow deriving from growth plate chondrocytes in zebrafish, although direct evidence will be needed to determine if any of these chondrocyte-derived lepr+ marrow cells behave as skeletal stem cells in zebrafish.”“In mice, lepr-Cre line in zebrafish, we cannot therefore conclude whether zebrafish lepr+ marrow cells are comparable to those described in mouse. In the future, the generation of new Cre lines to specifically mark lepr+ marrow cells in zebrafish will be needed to determine whether these chondrocyte-derived marrow cells also act as stem cells for osteoblasts and adipocytes in post-embryonic fish.”“Without a comparable long-isoform 5) The work on MMP9 is not strongly related to the rest of the manuscript. No direct evidence is offered to show that any of the phenotypes seen with the MMP9 loss of function relate to transdifferentiation of cartilage cells.mmp9 mutant data make several points of interest that relate to the manuscript. First, it establishes a similar genetic dependency of growth plate remodeling in zebrafish and mouse. Prior to our work, it was unclear whether mammalian-like growth plate remodeling operated in fish. Combined with the lineage tracing data presented, the similar mmp9 phenotypes further support conservation. In this revision, we have also performed additional mmp9 rescue experiments (increasing n from 2 to 8), which allow us to conclude that Mmp9 likely functions in chondrocytes for marrow fat production, thus providing additional supporting evidence for the conversion of chondrocytes into adipocytes .We feel that the 6) In Figure 6, how can it be excluded that the Brdu labeling observed is due to labeling of proliferating chondrocytes that have progressed to the hypertrophic stage by the time of visualization?We explain in the Materials and methods that fish were treated with BrdU for 1 hour followed by immediate fixation, hence detecting only cells that are actively proliferating or have only very recently divided. Progression of chondrocytes from the proliferative to the hypertrophic stage occurs on the order of days.


The incidence of Achilles tendon rupture in the UK is increasing and the best rehabilitation strategy for patients treated non-operatively remains unclear. We describe a statistical analysis plan (SAP) for the UK study of tendo Achilles rehabilitation (UK STAR) multi-centre randomised trial.UK STAR is a 1:1, multi-centre, parallel, two-arm, superiority randomised controlled trial. This study aims to evaluate the use of functional bracing compared to plaster cast for the management of acute Achilles tendon rupture in adult patients treated non-operatively. The primary outcome is the Achilles Tendon Rupture Score measured at 9 months after injury and will be estimated based on a linear mixed effects regression model adjusted for the stratification factor (centre) and other key prognostic variables. Secondary outcomes include complications, quality of life and resource use evaluated at 8 weeks and at 3, 6 and 9 months after the injury. Missing data will be summarised and reported by treatment arm. Full details of the planned analysis methods are described in this paper. Further study design details are published in the UK STAR protocol.The planned statistical analyses for UK STAR aim to reduce the risk of outcome reporting bias arising from prior data knowledge. Any changes or deviations from the current SAP will be described and justified in the final study report.ISRCTN62639639. Registered on 22 June 2016.International Standard Randomised Controlled Trial Number Registry,  Achilles tendon rupture affects over 11,000 people each year in the UK [.Several randomised controlled trials (RCTs) have evaluated the use of plaster cast and early movement and/or functional brace in the treatment of Achilles tendon rupture after surgical repair , 3. For UK Study of tendo achilles rehabilitation (UK STAR) is a randomised controlled trial  aiming tUK STAR is a pragmatic, multi-centre, parallel, two-arm, randomised controlled, superiority trial. Eligible patients are randomised 1:1, stratified by study centre using variable block sizes of 2, 4 and 6 in a 1:2:1 ratio via a secure, online randomisation system. Both plaster cast and functional bracing treatment techniques are routinely available at all recruiting sites. Stratification by centre helps to ensure any centre-related effect is equally distributed in the two trial arms. The patients and treating clinicians cannot be blind to the treatment as the type of rehabilitation used is clearly visible. The outcome data are collected and entered onto the trial central database from a paper form or via telephone by a research assistant/data clerk in the trial central office who is blinded to treatment arm to reduce the risk of assessment bias. Participants are also able to submit outcome data via an online questionnaire. Full details of the trial design, study population and study procedures have been published in the UK STAR protocol [The primary objective is to quantify and draw inferences on observed differences in the Achilles Tendon Rupture Score (ATRS)  between ▪ Quantifying and analysing observed differences in ATRS between the trial treatment groups at 8 weeks and at 3 and 6 months▪ Identifying differences in health-related quality of life between the trial treatment groups in the first 9 months▪ Determining the complication rate between the trial treatment groups in the first 9 months▪ Investigating, using appropriate statistical and economic analytical methods, the resource use, costs and comparative cost-effectiveness in the trial treatment groupsSecondary objectives include:The primary outcome is the ATRS measured at the 9-month follow up. The ATRS is a validated self-reported questionnaire , 9 consiQuality of life is measured by the EuroQol 5 Dimensions 5 Levels (EQ-5D-5 L) , a validComplications include re-rupture, blood clots/emboli, pressure areas and neurological symptoms in the foot. These are recorded from medical notes at the 8-week review when the cast/brace is removed and are patient-reported at 3, 6 and 9 months.The resource use data are collected at 8 weeks and at 3, 6 and 9 months and will be used to conduct a prospective health economic evaluation in the two trial arms.The ATRS collected at 8 weeks and at 3-month and 6-month follow up is a secondary outcome. Other secondary outcomes include quality of life, complications and resource use:The sample size calculation required 330 participants to be randomised. This assumed a population standard deviation (SD) of 20 points for the primary outcome ATRS at 9 months after injury  and a miThe trial reached its primary recruitment target of 330 participants prior to the end of the planned recruitment period, but recruitment was extended after consultation with the DSMC and Trial Steering Committee (TSC). The sample size was recalculated based on a larger population variability equivalent to 25 points SD, MCID 8 and 20% loss to follow up, which led to 516 participants, with a maximum limit set at 550 participants, to be recruited into the trial.A blinded analysis of data (not separated by treatment arm) will be undertaken prior to the final data lock in order to assess the distribution of variables, missing data distributions and outliers. This analysis will also be used to confirm the pre-specified key prognostic variables  to be included in the adjusted analysis and will also explore additional prognostic variables to be included in further supporting analyses. These will include smoking, diabetes mellitus and time since injury. The SAP will be updated following the blinded analysis and approved prior to the final data lock when the treatment code will be added to the database.p values to three decimal places are less than 0.05 (5% significance level).The primary analysis population will be intention to treat (ITT) and a complier average causal effect (CACE) population will be used as supporting analyses. The ITT population will include all randomised participants in their allocated groups and the CACE population will consist of randomised participants compliant with the intervention . PatientA Consolidated Standards of Reporting Trials for Patient Reported Outcomes  CONSORT. ParticiGenderAgeDays since injury; mechanism of injury; side of injuryHeight; weight; body mass Index (BMI)Smoking; cigarettes per day; number of years smokingAlcohol consumptionMedications; medical conditions, including diabetes mellitusATRS before injuryEQ-5D-5 L before injury and baseline after injuryCentreBaseline characteristics will include stratification factors and the following variables:Withdrawals and losses to follow up, together with reasons, will be reported by treatment arm and compared in each of the periods leading up to the 8-week and 3-month, 6-month and 9-month time point. In the event of a tendon re-rupture following which the participant undergoes surgery, the participant will not be treated as lost to follow up. Differential losses between the groups will be compared and deaths (and their causes) will be reported separately.The patterns of data availability for primary and secondary outcomes from baseline to end of follow up and reasons for missingness, where known, will be summarised in the two treatment groups . The nature and pattern of missing data (missing completely at random (MCAR), missing at random (MAR) or missing not at random (MNAR)) will be explored.The primary outcome ATRS is expected to have low levels of item missingness. Validation rules will ensure that data are entered in the correct format, within valid ranges, minimising the chance of missing data. Where ATRS items are missing and a minimum of 5 out of 10 ATRS items are present, a pro-rata estimation of the ATRS score will be imputed based on the average  of the available ATRS item responses for the primary analysis. The extent of pro-rata estimation will be reported for each treatment group.Missing continuous primary and secondary outcomes will be handled as part of the likelihood-based estimation of the linear mixed model in the primary analysis where the MAR assumption holds, allowing sufficient generality to include baseline variables thought to be important predictors . SensitiOne group of patients will receive a functional brace  after randomisation and one group will receive a plaster cast. The compliance with treatment will be reported by intervention group and summarised where possible with reasons for not receiving the assigned treatment. A CACE analysis  will be The primary outcome, ATRS at 9 months after randomization, will be summarised on an ITT basis as mean and SD in each of the two treatment groups, functional brace and plaster cast, assuming the data are approximatively normally distributed. The main findings of the trial will be reported as the difference in the ATRS between the two treatment arms, estimated with a linear mixed effects regression model, including outcome information from all follow-up points and adjusting for centre, age, gender and baseline ATRS. Supporting analyses will also be reported, including other important prognostic variables as identified during the blind review. As individual clinicians will treat only a small number of patients, important clinician-specific effects are not expected, but recruiting centre will be included in the model as a random effect factor to adjust for potential cluster differences. Estimates of treatment effects will be presented with 95% CIs.Where severe departure from normality in the difference in ATRS between the two groups is identified , the first approach to attain normality will be data transformation or the use of a different metric such as change from baseline. If the data cannot be transformed to reflect normality, then the Mann-Whitney U test will be used  and the medians and IQRs will be reported in each treatment arm.t test, based on a normal approximation for the ATRS score. Estimates of treatment effects will be presented with 95% CIs for both adjusted and unadjusted analyses. The ITT adjusted analysis of the primary outcome will be used to determine the success or otherwise of the trial. Sensitivity analysis to confirm the robustness of the ITT primary analysis conclusions will be repeated in the CACE population and using multiple imputation, where appropriate.Differences between treatment groups will be analysed unadjusted using the Student t test will be presented together with their associated 95% CI. The EQ-5D-5 L will also be analysed in this way.To explore recovery in the two treatment groups over time, the ATRS will be analysed further. This will summarise the longitudinal data collected at all four time points to a single value, the area under the curve (AUC)  and willp values for comparison between the two treatment groups.Continuous secondary outcomes will be analysed on an ITT basis using the methodology described for the primary outcome, and results will be reported at each intervening time point. Complications in each treatment arm will be explored and analysed on an ITT basis, reported as numbers (with percentages) and compared at 8 weeks and at 3, 6 and 9 months using Fisher’s exact or chi-squared test. Results will be reported with their associated 95% CI and Some adverse events and serious adverse events (SAEs) are foreseeable as part of the proposed treatment and are recorded as complications. Unexpected but related SAEs and suspected unexpected serious adverse reactions (SUSARs) that occur between date of consent and the 9-month follow-up point will be reported and compared between the functional bracing and plaster cast groups in the ITT population by examination of 95% confidence intervals for differences in incidence.There are no planned subgroup analyses for any of the follow-up primary or secondary outcomes.A separate heath economic and cost-effectiveness analysis will be conducted and details of this analysis described in a separate health economics analysis plan.All analysis will be carried out using appropriate validated statistical software such as STATA, SAS, SPLUS or R. The relevant package and version number will be recorded in the statistical report.The UK STAR trial will provide functional outcome data on two rehabilitation strategies, functional brace versus plaster cast, in non-operatively managed patients with a rupture of the Achilles tendon. One study limitation is that it will not be possible to blind patients to their allocated treatment, as the plaster cast or functional brace will be clearly visible.This paper provides details of the planned statistical analyses for UK STAR and any changes from the current protocol or SAP will be described and justified in the final report. The aim of the pre-specification of the analysis is to reduce the risk of outcome reporting bias and data-driven results .Recruitment into the trial opened on 1 August 2016 and closed on 31 May 2018. A total 541 participants were recruited from 39 sites. Follow up for the trial outcome data will be completed by March 2019, which will then be followed by the analysis.