Chimeric pestiviruses based on CSF virus (CSFV) and the related bovine viral diarrhea virus (BVDV) have been licensed as live marker vaccines in Europe and Asia, but cross-reactive antibodies can cause problems in DIVA application due to close antigenic relationship. To develop marker vaccine candidates with improved DIVA properties, three chimeric viruses were generated by replacing Erns of CSFV Alfort-Tübingen with homologue proteins of only distantly related pestiviruses. The chimeric viruses “Ra”, “Pro”, and “RaPro” contained Erns sequences of Norway rat and Pronghorn pestiviruses or a combination of both, respectively. In porcine cells, the “Pro” chimera replicated to high titers, while replication of the “Ra” chimera was limited. The “RaPro” chimera showed an intermediate phenotype. All vaccine candidates were attenuated in a vaccination/ challenge trial in pigs, but to different extents. Inoculation induced moderate to high levels of neutralizing antibodies that protected against infection with a genetically heterologous, highly virulent CSFV. Importantly, serum samples of vaccinated animals did not show any cross-reactivity in a CSFV Erns antibody ELISA. In conclusion, the Erns antigen from distantly related pestiviruses can provide a robust serological negative marker for a new generation of improved CSFV marker vaccines based on the chimeric pestivirus concept.Classical swine fever (CSF) is one of the most important viral diseases of pigs. In many countries, the use of vaccines is restricted due to limitations of subunit vaccines with regard to efficacy and onset of protection as well as failure of live vaccines to  Pestivirus within the family Flaviviridae. CSF is endemic in many pig-rearing countries worldwide and causes tremendous economic losses [Classical swine fever (CSF) is one of the most important viral diseases in pigs. Causative agent is the classical swine fever virus (CSFV), an RNA virus belonging to the genus c losses . These lc losses .differentiate infected from vaccinated animals (DIVA principle). The viral glycoprotein Erns is a promising target to establish a discriminating principle based on antibodies. Alternatively, it has been demonstrated in experimental vaccines that deletion of single, highly conserved epitopes in the E2 protein can provide a serological negative marker [In many countries, the use of vaccines, even in emergency situations, is restricted due to limitations of available subunit vaccines with regard to efficacy and onset of protection or due to failure of conventional live vaccines to e marker . The conrns, was designed based on the live attenuated CSFV vaccine strain LOM. In this CSFV chimera, the complete Erns and signal peptide encoding sequence was replaced by corresponding BVDV sequences [Different MLV candidates with marker properties have been developed applying modern molecular techniques, but until now, only two have been licensed. In Europe, a chimeric pestivirus, named CP7_E2Alf, was licensed as a marker MLV in 2014. It harbours the E2 coding region of CSFV in the context of the bovine viral diarrhea virus (BVDV) strain CP7 . This chequences . The vacequences . In Chinequences . No infoin vitro and in vivo. A first animal trial in pigs demonstrated that chimeric vaccine strains expressing only distantly related Erns antigens are an interesting option to provide both an efficient protection against CSF and a robust serological negative marker with a considerably reduced risk to induce cross-reactive antibodies that may interfere with CSF diagnosis.The genetic construction of chimeric pestiviruses has been demonstrated to be one promising strategy for generation of efficient MLV with marker properties. In principle, chimerization of closely related viruses enables to maintain efficient viral replication, but at the same time may be problematic in terms of induction of cross-reactive antibodies. The discovery of pestiviruses being only very distantly related to CSFV provides new options to design improved CSF marker vaccine candidates based on the chimeric pestivirus concept. In the present study, several chimeric CSFV were designed, which differed in their replication properties rns encoding sequence of a moderately virulent CSFV by the homologous sequences from pestiviruses distantly related to CSFV. A “Ra” chimera (CSFV-2697) containing the Erns sequence of Norway rat pestivirus (NrPV) isolate NYC-D23 (GenBank access. No. NC025677), a “Pro” chimera (CSFV-2677) harbouring the Erns sequence of Pronghorn antelope pestivirus (GenBank NC024018.2), and a “RaPro” chimera combining parts of both Erns sequences (CSFV-2695) were generated (rns encoding sequence are derived from NrPV followed by 186 nucleotides (position 1660–1845) of the Pronghorn pestivirus sequence. The chimeric pestivirus vaccine candidates were generated on basis of the infectious cDNA clone pAlfort-p447 of CSFV strain Alfort-p447 [. The sequence of the cDNA plasmid used in this study (pAlfort-p2477) is almost identical to the sequence previously published for CSFV Alfort-p447, which contains two additional guanine residues at its 5′ genome end (GenBank LT593760). In addition, pAlfort-p2477 is harbouring three synonymous mutations  to generate artificial BsiWI and KasI restriction sites, respectively. To produce synthetic viral RNA genomes, pAlfort-p2477 was linearized using a SmaI site located at the 3′ end of the CSFV sequence. The sp6 promoter sequence located directly upstream of the CSFV specific sequence was used for in vitro transcription. Synthesized RNA was electroporated into SK6 cells as described previously [To develop improved CSFV marker vaccine candidates, three chimeric variants were generated by replacing the Eenerated . In the ort-p447 ,15. The position 60–1845 oeviously . For viseviously .Figure rns sequence. The attenuation control group was not challenged. Challenge infection of the other groups was performed with the genetically heterologous CSFV strain Koslov (genotype 1.1) which has been often used in vaccination/challenge trials due to its extraordinary high virulence. The unvaccinated control group  consisted of three pigs to demonstrate that the virus stock of CSFV strain Koslov used for challenge infection is able to induce severe disease under the experimental conditions. As CSFV Koslov usually leads to death of all inoculated pigs within less than one week, the experiment was stopped at 12 and 13 days post challenge (dpc). Post-mortem investigation included the visual control of all organs for signs typically seen as consequence of CSF and sampling of tissues, including kidney, spleen, mandibular lymph nodes and parotic gland.The animal experiment consisted of five study groups comprising twenty-three 10 weeks old hybrid pigs obtained from a commercial farm. Three study groups of five pigs were inoculated with the vaccine candidates “Ra”, “Pro”, and “RaPro”, respectively. One additional group of five pigs inoculated with Alfort-p2477 served as attenuation control to be able to monitor the extent of attenuation of the chimeric vaccine candidates depending on the inserted EPigs were clinically examined and rectal body temperature was measured daily. Clinical scores were calculated as described earlier  based onFor detection of CSFV genomes, RNA was isolated from medium of re-suspended fecal and oral swap samples or EDTA-blood after one freeze–thaw cycle using the QIAamp Viral RNA Mini Kit . For RNA isolation from tissue samples a NucleoSpin RNA Kit (Macherey/Nagel) was used as recommended. To determine the CSFV genome loads quantitatively, an accredited CSFV specific real-time PCR was applied using a QuantiTect Probe RT–PCR mastermix  with primers and probe previously established .rns- specific antibodies the IDEXX CSFV Ab Test  and the pigtype CSFV Erns Ab ELISA kits  were used according to the manufacturer protocols, respectively. For quantification of neutralizing antibodies (nAbs), three independent titrations (each in duplicates) were performed with the parental CSFV strain Alfort-p2477 (genotype 2.3) and the heterologous challenge virus strain Koslov (genotype 1.1) according to the procedure established at the EU and OIE Reference Laboratory for CSF [For detection of CSFV E2- and E for CSF .rns encoding sequence in the cDNA of CSFV Alfort-p2477 was confirmed by PCR and subsequent nucleotide sequencing. Transfection of in vitro transcribed RNA into porcine SK6 cells resulted in expression of viral proteins as shown by immunofluorescence testing. The “Pro” chimera completely infected a porcine cell culture monolayer like the parental CSFV strain Alfort-p2477. Further analysis revealed that the “Pro” chimera replicated to titers comparable to Alfort-p2477 .Successful replacement of the original Ert-p2477 . The “RaCID50/ml . DetermiTo characterize the properties of the chimeric viruses in the natural host, an animal experiment was performed (6.7 TCID50/pig) of the most virulent CSFV strain available, none of the vaccinated animals showed fever or any other signs of disease after challenge infection /ml blood were reached at ten days post i.m. application. Interestingly, two animals inoculated with the “Pro” chimera showed a simultaneous increase, however, reaching lower genome loads (107−108 GE/ml). Genome levels decreased from 10 dpv on, but both animals were still genome positive at 28 dpv. Although not showing any clinical signs of CSF, these two pigs were the same animals that showed initial increase of body temperatures. The other three animals of the “Pro” vaccinated group were genome positive in the blood samples only at days 7 and 10 and with lower genome loads ranging between 105 and 106 GE/ml. All five pigs of the “Pro” inoculated group transiently shed viral genomes via saliva between 7 and 21 dpv, but only the two pigs with the long term viremia also shed viral genomes with the feces. In the “RaPro” vaccine study group low amounts of viral genome were detectable in the blood of only three animals. No genome was detected in any of the oral and fecal swap samples. All five animals inoculated with the “Ra” chimera were tested genome negative in blood, saliva and feces, which is consistent with the low viral replication observed in cell culture.To investigate the potential of the vaccine candidates to cause viremia and to be excreted, a real-time RT–PCR was used for viral genome detection and quantification  were first detectable between 14 and 21 dpv and homologous titers ranged between 147 and 649 ND50/ml (28 dpv). In the “Ra” vaccination group, nAbs were detected as early as 10–14 dpv, but titers were slightly lower in average reaching 113–320 ND50/ml (homologous virus) and 15–53 ND50/ml (heterologous virus). The “RaPro” chimera induced nAb between 10 and 21 dpv. The average titers ranged between 347 and 587 ND50/ml (homologous virus) and 67–180 ND50/ml (heterologous virus) and thus were highest among the vaccinated groups. After challenge infection, a dramatic increase of titers was observed in all vaccinated pigs. At 12/13 dpc  average titers were determined highest in the “Ra” vaccination group , lowest in the “Pro” vaccination group , while intermediate neutralization titers were detected in animals vaccinated with the “RaPro” chimera  and the genetically heterologous challenge strain Koslov (genotype 1.1)  ELISA for detection of CSFV specific Ab  was reported to occur already after 1 week and partial protection against a moderately virulent strain already as early as 2 dpv [rns) or vice versa (CP7_E2Alf). At least for the latter it has been shown in different studies that cross-reactive Abs are induced that interfere with a robust negative serological marker for DIVA [rns vaccine are lacking. Cross-reactivity is likely to occur, although not reported in a study investigating seroconversion in a limited number of animals [The presented study was performed to design and characterize MLV candidates with improved marker properties. For this purpose, chimerization of CSFV with only distantly related pestiviral sequences was performed. The replication properties of the three generated chimeric viruses as 2 dpv ,23. Earlfor DIVA ,24,25. C animals . The use animals . It willrns encoding sequences of only distantly related pestiviruses probably results in a non-functional genome or a poorly replicating virus. This assumption is in line with the finding of a previous patent study (WO2017114778A1). The inventors claimed that replacement of the 3′ region of an Erns gene from a pestivirus that is genetically closely related to the mutant pestivirus will be required to achieve a well replicating chimera. In the presented study, it was demonstrated that the CSFV genome can tolerate the replacement of the Erns encoding sequence by only distantly related sequences. Replacement of the complete CSFV Erns by the Pronghorn Erns resulted in a very well replicating chimeric virus. In a previous study, chimerization of the genome of BVDV strain NADL with the Erns coding sequence of the Pronghorn pestivirus led to reduced replication compared to the parental strain and chimera containing the Erns of the more closely related giraffe and reindeer pestiviruses, respectively [rns sequence is the genetically most distinct sequence used to design pestivirus MLVs with marker properties. Low similarity and limited compatibility with the CSFV backbone may explain that the chimera replicated poorly in cell culture. However, it was possible to improve in vitro replication by replacing the 3′ portion of the NrPV pestivirus Erns encoding sequence by the homologous sequence of Pronghorn pestivirus. The observation that in vitro replication of the “RaPro” chimera increased after passaging on cell culture is probably due to acquisition of adaptive mutations. Occurrence of mutations during cell culture passaging is a helpful mechanism to improve replication of artificially constructed genomes of RNA viruses. In this line, multiple mutations have been previously described in the CP7_E2Alf chimera, including one important amino acid change in the Erns reported to be responsible for efficient replication in porcine cells [rns are different to the mutations observed in CP7_E2Alf and another mutation known to be important for acquired heparan sulfate binding during cell culture adaptation [Starting point of the presented study was the hypothesis that genetically distinct pestiviruses might open an avenue to create replicating chimeric viruses with improved marker properties due to significantly reduced risk of inducing cross-reactive Ab. In the past, numerous experimental CSF marker vaccine candidates have been generated and evaluated, all of them based on sequences of closely related pestiviruses . It can ectively . So far,udy WO201114778A1.aptation . Of notein vitro, but also in vivo. Virus genomes of other MLVs can consistently be detected in tonsils up to day 42 and in blood samples within the first 14 dpv by real-time RT–PCR [Although all three chimeric viruses were attenuated, the introduction of heterologous sequences into the CSFV Alfort-p2477 backbone had different impact on the replication properties not only e RT–PCR . Thus, ae RT–PCR .rns Ab based DIVA assay. Further studies, including a higher number of vaccinated animals and repeated vaccination, would be necessary to determine differences in the robustness of the three introduced serological negative markers. Due to higher antigenic distance to CSFV Erns, the NrPV and “RaPro” Erns antigens can be expected to be superior in serological DIVA application compared to the Pronghorn pestivirus Erns.Although high amounts of CSFV Ab were induced, sera of all vaccinated pigs – independently on the applied vaccine candidate – revealed to be non-reactive in the CSFV Erns encoding sequences allows designing chimeric attenuated viruses that can be propagated in amounts suitable for vaccine production. Chimeric MLVs containing Erns encoding sequences of distantly related pestiviruses are a very promising strategy for establishing an improved serological negative marker in CSF marker vaccines.Taken together, combination of CSFV genomes and genetically distinct EClick here for additional data file.


Prior ex vivo histological postmortem studies of autism spectrum disorder (ASD) have shown gray matter microstructural abnormalities, however, in vivo examination of gray matter microstructure in ASD has remained scarce due to the relative lack of non-invasive methods to assess it. The aim of this work was to evaluate the feasibility of employing diffusional kurtosis imaging (DKI) to describe gray matter abnormalities in ASD in vivo. DKI data were examined for 16 male participants with a diagnosis of ASD and IQ>80 and 17 age- and IQ-matched male typically developing (TD) young adults 18–25 years old. Mean (MK), axial (AK), radial (RK) kurtosis and mean diffusivity (MD) metrics were calculated for lobar and sub-lobar regions of interest. Significantly decreased MK, RK, and MD were found in ASD compared to TD participants in the frontal and temporal lobes and several sub-lobar regions previously associated with ASD pathology. In ASD participants, decreased kurtosis in gray matter ROIs correlated with increased repetitive and restricted behaviors and poor social interaction symptoms. Decreased kurtosis in ASD may reflect a pathology associated with a less restrictive microstructural environment such as decreased neuronal density and size, atypically sized cortical columns, or limited dendritic arborizations. Individuals with ASD have been found to have an array of complex brain abnormalities including early overgrowth3, microstructural disorganization4, and deficits in both cytoarchitecture5 and functional connectivity6. In vivo studies of microstructural abnormalities in ASD have focused primarily on white matter8, leaving the nature and distribution of gray matter (GM) abnormalities largely uncharacterized.Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by a range of social, executive functioning, and sensory deficits9 and decreased cell size in the cingulate cortex10 and frontal lobe regions4, along with heterotopia and decreased dendritic branching in frontal and temporal gyri11. Ex vivo research has also shown increased markers of inflammation in the anterior cingulate cortex and middle frontal gyrus12, along with under-developed minicolumn formation across the cortex15. Although ex vivo studies have been essential for understanding the underlying cellular pathology of ASD, the literature is notably variable likely due to the heterogeneous nature of the disease and the inevitable limitations of postmortem research, which is often based on highly inhomogeneous and small samples ranging from 1 to 11 ASD subjects17, and typically focuses on isolated brain regions. Thus, techniques for examining GM microstructure in vivo would allow whole-brain analysis within larger, well-matched samples. Such techniques are necessary for better describing the location, degree and developmental course of GM microstructural pathology and their variation across individuals affected with ASD.Postmortem studies of ASD have implicated several abnormalities in cortical GM. Early studies found increased cell packing, particularly in the prefrontal cortex21. The advent of high angular resolution multi-shell diffusion imaging methods has allowed extending the in vivo investigation of microstructure to gray matter23. Among these new methods, diffusional kurtosis imaging (DKI) was the first to show high sensitivity to GM changes due to development in typical children and adolescents as well as to document atypical development in attention-deficit hyperactivity disorder (ADHD), a childhood disease24. DKI improves upon the DTI characterization of the diffusion signal in tissues by including an additional directional metric, the kurtosis index, to describe non-gaussian diffusion contributions to the MR signal25. Non-Gaussian diffusion contributions are inherent to biological tissues such as the brain and arise from the complex organization of microscopic membranes, organelles, and neuronal compartments25. Of note, kurtosis metrics have been shown to be able to describe complex structure in the absence of anisotropy26. Increased kurtosis is assumed to describe increasingly restricted and complex environments and has been shown to characterize brain development and maturation24, as well as pathological features leading to increased complexity 29. Decreased kurtosis is likely indicative of tissue disintegration and shrinkage32. DKI provides both a mean kurtosis (MK) metric, which characterizes kurtosis averaged across all spatial directions, and, like DTI, two directional metrics: axial kurtosis (AK), which quantifies kurtosis along the primary or largest diffusivity direction and radial kurtosis (RK), which measures the averaged kurtosis in the plane perpendicular to the primary diffusivity direction. The use of directional kurtosis metrics in GM is supported by recent findings indicating highly directional diffusion in the cortex aligning either radially or both radially and tangentially to the cortical ribbon33.Diffusion-weighted MRI (dMRI) is a powerful tool for describing microstructural tissue organization at micrometer scale by probing water molecule displacement, which is sensitive to microscopical barriers  and organelle organization  within each voxel. To date, diffusion tensor imaging (DTI), which approximates water diffusion in tissue using a Gaussian distribution, has remained the most widely used diffusion technique. DTI associated indices, fractional anisotropy (FA), and mean (MD), axial (AD), and radial (RD) diffusivities have been widely applied to characterize anisotropic white matter and have been found to be sensitive to a number of white matter pathological changes such as dysmyelination, axonal loss, and inflammation36 and in several conditions including Parkinson’s disease37, chronic pain38, multiple sclerosis39, traumatic brain injury40, multiple sclerosis39, schizophrenia41, Alzheimer’s disease42 and attention-deficit/hyperactivity disorder43. Ex vivo studies investigating GM changes in several rodent neurodegenerative models have documented that increased kurtosis parallels increased fiber dispersion29, protein accumulation27, and astrogliosis28, and that decreased kurtosis co-occurs with cytotoxic edema44, abnormal neurofilaments, and axonal shrinkage and loss45. In addition, directional kurtosis estimates have been found to detect unique characteristics of tissue compared to MK in both healthy and clinical samples46. Therefore, we hypothesized that mean and directional kurtosis in the GM of ASD patients is altered as a result of one or more cellular pathological processes observed in ASD: disorganization and alterations of cortical laminar cytoarchitecture and dendrites, decreased cell size, increased cell packing, gliosis, and narrow minicolumn formation47.To date, DKI has been shown to be sensitive to GM microstructural change in the aging brain50. An initial study found slightly increased MD in total brain GM in adolescents with ASD ages 12–18 years old48, while another study employed a Restriction Spectrum Imaging (RSI) approach and reported significantly reduced gray matter neurite density (ND) in the left fusiform cortex of adolescents with ASD ages 7–17 years old at multiple-comparison correction level49. This second study additionally found slightly increased MD in the parietal and occipital lobes, but this finding did not survive multiple-comparison correction49. Most recently, reduced orientation dispersion (ODI) and ND were found in ASD patients with impaired recognition of facial emotional expressions in the occipital lobe, fusiform gyrus, and inferior parietal and superior temporal regions using the Neurite Orientation and Dispersion Imaging (NODDI) approach50. Reduced ND in ASD was hypothesized to be related to the ex vivo finding of a lower density of, or thinning of, myelinated axons in the gray matter cortex, while decreased ODI was postulated to be associated with the loss of dendritic spines49. The increased MD noted in two of the studies was proposed to be a result of increased water due to the inflammatory response often reported in ASD49. This previous work has suggested that dMRI may be able to distinguish different GM microstructural pathologies in ASD.To date, only few studies have investigated GM cortical microstructure in ASD using in vivo dMRI methods, gray matter cortical differences in a group of young adults with ASD versus age, sex and IQ-matched typically developing (TD) control participants and to test the hypothesis that kurtosis is associated with the severity of core ASD symptom deficits . Compared to biological model-based multi-shell dMRI methods, such as NODDI, DKI is a data driven mathematical model that makes no assumptions regarding underlying microstructure, and thus is independent of such assumptions, which may not be valid in various pathologies. Moreover, compared to DTI metrics, DKI metrics have been shown to be robust with respect to partial volume averaging with cerebro-spinal fluid51, an artifact likely to at least partially affect cortical regions.In this study, we employed DKI to characterize, non-invasively and in vivop = 0.678), education (p = 0.463), or full-scale IQ score (p = 0.143)  Table ; Fig. 2.No differences in AK were found between groups in either lobar or sub-lobar regions.In the ASD group, increased MK was associated with fewer repetitive and restrictive behaviors and better social interaction, with only the left entorhinal cortex’s association with repetitive and restrictive behaviors and the right parietal lobe’s association with social interaction reaching significance after correction for multiple comparisons Table ; Fig. 3.17, while narrower cell minicolumns with decreased neuropil in the GM of patients with ASD have been extensively documented16. These findings suggest reduced microstructural complexity in the GM of the ASD brain, consistent with our findings of decreased MK and RK. Further supporting this hypothesis, rodent studies have reported that decreases in MK may reflect neuronal loss, microgliosis, myelin disruption31, neuron disorganization and cytotoxic edema44, with decreases in both MK and RK reflecting reduced GM neurite density53.The data presented here provides, for the first time, in vivo evidence of GM microstructural pathology in ASD by employing DKI to non-invasively probe tissue organization and complexity. Significantly decreased cortical kurtosis and diffusion noted in the ASD group may reflect one or several pathological processes reported by previous histological studies. The majority of ex vivo studies on ASD found decreased cell size and limited dendritic arbors25. In the data investigated here, RK, a DKI-specific parameter of diffusional kurtosis perpendicular to the primary direction of diffusion, was the most sensitive metric in quantifying between-group differences, highlighting areas with known cellular pathology in ASD  compare in identifying GM microstructural pathology in ASD across the lifespan. The slightly increased MD values in total brain GM and in the parietal and occipital lobes reported by these previous studies, which however did not pass multiple corrections level, are somewhat contradictory to our findings of significantly decreased MD in the right posterior cingulate cortex49. More research will be important to elucidate where and to what extend MD is altered in varying ASD populations.Compared with the recent dMRI work investigating GM cortical microstructure in ASD using other methods than DKI65 up Table ; Fig. 3.9, decreased cell size and dendritic branching10 and under-developed minicolumn formation15. However, they are inherently limited by their invasive nature and their findings are difficult to translate into clinical practice and personalized medicine. Advanced dMRI imaging, which is non-invasive and can be acquired in a clinically feasible time frame and setting, may be used to extend and refine these decades-long documented microstructural abnormalities in ASD. Of note, dMRI has mostly been applied to detect in vivo ASD white matter pathologies despite GM showing a larger range of histological abnormalities that may be useful clinical biomarker targets. For example, reduced synaptic density in GM has recently been used as a radiopharmaceutical target in schizophrenia74. Kurtosis is an index of microstructural complexity, which in addition to potentially detecting an array of histological changes in ASD it has both successfully revealed the presence of isolated microstructural changes in preclinical models75 and been superior to conventional macrostructural MRI in differentiating patients in other neurological disorders41. Therefore, although this pilot study primarily aimed to test the ability of DKI to detect GM microstructural abnormalities in ASD and their relationship to symptoms, its results provide support for future larger DKI studies that may better track, predict and treat ASD deficits.Postmortem studies have provided valuable information regarding the underlying GM microstructural pathologies of ASD, most notably reporting increased cell packingSeveral limitations of this study need to be considered. First, the sample size was relatively small, and the study had a purely cross-sectional design. In part, our limited sample size stems from our decision to select a relatively homogeneous group by studying males with ASD within a limited age range (18–25 years old) and without intellectual disability (IQ < 80), which we believe strengthened our ability to detect group differences. However, it will be important to replicate these results in larger samples and to examine gray matter in both sexes and across larger age ranges. Second, these data were acquired before the availability of higher resolution diffusion methods that may reveal more accurate diffusion measurements. Third, two different T1-weighted (T1w) MRI protocols were used for the within-subject atlas segmentation and ROI analysis. Paired t-tests showed that different T1w acquisition had no significant effect on the calculation of MK or RK metrics but had some minimal effects on MD metrics and therefore the MD results presented here should be interpreted cautiously 77 was administered to obtain Full Scale intellectual quotient (FSIQ) scores and confirm the lack of intellectual disability. Handedness was obtained using the Chapman and Chapman handedness questionnaire78. The study was approved by the institutional review board at the NYU School of Medicine and performed in accordance with all National Institute of Mental Health (NIMH) and NYU School of Medicine relevant guidelines and regulations. For this study, 17 ASD patients and their parents provided informed consent. For another 9 ASD participants, for whom parents were not available for participation, competence to provide consent was assessed at the beginning of the visit by trained personnel. All these study participants were adults deemed able to provide informed consent and provided informed consent before participation according to our institutional rules.A total of 26 male individuals with a potential ASD diagnosis and 36 age-matched male TD participants were recruited by advertisement within the community and through autism organizations in the New York City metropolitan area. Diagnoses were confirmed by administering the Autism Diagnostic Observation Schedule (ADOS)Subjects whose imaging data displayed significant motion artifacts and for whom repeated data acquisition was not successful were not included in the study. A neuroradiologist examined all scans for gross brain abnormalities and to exclude any subjects suspected of an organic brain disorder.To match the two groups on IQ, TD subjects underwent first only a review of medical history and the IQ assessment. As new ASD participants were added to the study, TD participants were invited for imaging if they had IQ values similar to the recruited ASD participants. Twelve TD participants were either no longer available to participate for imaging by the time they were invited or had not matched any of the ASD participants and were thus dropped from the study. In addition, seven other TD participants met exclusionary criteria, which included a previous or current diagnosis of attention deficit hyperactivity disorder (2 participants), leukemia (1 participant), or MRI findings (4 participants). Among the potential ASD participants, two were excluded due to IQ < 80, three since they did not meet the threshold criteria for an ASD diagnosis, and one due to the presence of MRI contraindications. Two additional ASD participants requested to terminate the MRI procedure before any meaningful data could be acquired. Finally, two additional ASD data sets were not used in this study due to poor image quality. The remaining data sets (17 TD and 16 ASD participants) were included in the analyses  with 12 non-collinear encoding directions acquired for b = 1000 s/mm2, and 42 non-collinear encoding directions and for b = 2000s/mm2. Ten non-weighted diffusion images (b = 0 s/mm2) were also collected. Diffusion data acquisition was repeated twice. To correct for image distortions from B0 field inhomogeneities phase and magnitude field map images were acquired coplanar to the diffusion acquisition using the Siemens product sequence with echo-times of 8 ms and 10.46 ms.All MRI data were acquired on a 3T Trio MRI . Images were acquired using a body coil for transmission and a 12-channel array coil for reception. Diffusion imaging data were acquired using a twice-refocused diffusion-weighted echo planar imaging (EPI) sequence with a GRAPPA parallel imaging factor 2, and 24 reference lines. Between 55 and 60 slices were acquired using an isotropic voxel size of 2.3 × 2.3 × 2.3 mm3 isotropic voxel size was collected for 8 participants , and b) a MPRAGE with a 160 × 240 × 256 matrix and a 1 × 1 × 1.2 mm voxel size was collected for 25 participants . Eleven participants (9 ASD and 2 TD) had both T1w acquisitions with the rest of the participants having only one MPRAGE image.In addition to the diffusion data, T1w images were acquired using a magnetization prepared rapid gradient-echo (MPRAGE) sequence and used in the atlas registration procedure for ROI analyses, and additionally for clinical evaluation of gross brain abnormalities in each subject. Two T1w protocols were used: a) an axial MPRAGE with a 192 × 256 × 188 matrix and a 1 mmhttp://www.fmrib.ox.ac.uk/fsl)79. Data pre-processing steps included: (1) correction of B0 field inhomogeneities using the field map and FSL fugue and prelude functions; (2) image smoothing with a three-dimensional Gaussian filter with σ = 1.2 mm; (3) visual inspection of images for signal dropouts, blurring from movement and removal of artefactual images, and; (4) adjustment of the encoding gradients’ matrix for rotations during the motion correction step. After image correction, diffusion and kurtosis tensors were calculated as previously described in the field80 and employed to calculate three-dimensional maps of MK and MD. Additionally, AK and RK maps were obtained to test for directional differences in kurtosis microstructural properties.Data were preprocessed and images were corrected for motion and distortions from eddy currents along with magnetic field inhomogeneities using in-house developed code in Matlab , Interactive Data Language , and FMRIB Software Library  was used to construct the cortical surface of each participant based on the high-resolution T1w image. The FreeSurfer pipeline for surface construction, processed the images by segmentation of tissue types, tessellation of the grey/white matter junction, inflation of the folded surface and parcellation by the Desikan–Killiany atlas81. Each subject-specific cortically-labeled volume was then warped into dMRI space using FreeSurfer’s rigid-body transformation81. Mean diffusion metrics were obtained for each of the four regional cortical lobes and 68 cerebral cortex GM ROIs for each subject and used to conduct group comparisons.FreeSurfer  and minimal effect on MD metrics with no overlap between the ROIs affected by the T1W protocol and those shown to have significantly different MD in between-group comparisons  we conducted paired t-tests on data from 11 participants  that had both T1w acquisitions. For each of the 11 participants, image processing and registration to diffusion space was done separately for both T1w acquisitions and dMRI metrics were subsequently compared in the 68 ROIs delineated by the Desikan–Killiany atlas82 using SPSS 20.0 . Although the two groups did not statistically differ in demographics  tests controlling for age to first compare diffusion metrics  across the four anatomically defined cortical lobes , and then secondarily across the 68 cortical sub-lobar GM ROIs delineated by the Desikan–Killiany atlascs Table , DKI metPearson’s and Spearman’s correlations were used to explore the relationships between diffusion metrics that were found to show significant between-group differences  and ADI-R domain scores in ASD.52 to control the False Discovery Rate at 5%. Multiple comparison correction was applied first to (1) the 8 bilateral GM lobes, and then to (2) the 68 sub-cortical GM ROIs delineated by the Desikan–Killiany atlas. The Benjamini–Hochberg multiple comparison correction was performed on a single dMRI variable  at a time. Individual results were considered significant at BH q ≤ 0.05. Differences at p ≤ 0.05, uncorrected were considered to only reach trend-level and are listed in the tables and in the supplementary material.To account for multiple comparisons across the four cortical lobes and 68 cortical regions, we employed the Benjamini–Hochberg (BH) procedureSupplementary Tables.


The understanding of pain pathophysiology is continuously evolving. Identifying underlying cellular and subcellular pathways helps create opportunities for targeted therapies that may prove to be effective interventions. This article is an update on four areas of developing knowledge as it pertains to clinical management of patients with pain: nerve growth factor antagonists, microglial modulation, AMP-activated protein kinase activators, and genetic pain factors. Each of these areas represents novel targets for targeted therapies to prevent, treat, and modify the disease course of acute, chronic, and neuropathic pain. Currently most pain management techniques do not target these pathways directly, but there is promising evidence to suggest that the field is advancing toward available therapies in the near future. New discoveries in the mechanisms of persistent pain pathophysiology are evolving areas of research and developments of target-specific therapies. Conversely, the intricate interplay of multiple cellular and subcellular components and the ubiquitous presence of these components on non-nociceptive structures make treatments complicated by side effects.Nonetheless, this review focuses on four evolving areas of pain research that show promise in effectively identifying and treating persistent pain. This is not an exhaustive list of ongoing research; these topics were selected to represent a cross section of various fields of study that enhance our understanding of pain physiology and have identified possible therapeutic targets. Though trials are underway in some of these areas, many are developmental and require further study before any agents are available for clinical use.This article briefly introduces the state-of-the-art progression in nerve growth factor antagonists, microglia actions in the central nervous system, AMP-activated protein kinase (AMPK) activators, and genetic alterations affecting pain perception and treatment.Further exploration of these topics can be reviewed by referring to the references included at the end of this article.1Nerve growth factor (NGF) is a neurotropin implicated in inflammation and sensitization phenomenon. Increased concentrations have been found in tissues undergoing an inflammatory response. Biochemically, it is a polypeptide that binds to tropomyosin receptor kinase A (TrKA) and p75 receptors. TrKA receptors on mast cells trigger histamine release, potentiating the inflammatory pathway and eventual pain hypersensitivity. Activation of TrKA and P75 receptors on peripheral nociceptors results in downstream neurotransmitter cascade and increased transcription of various neurotransmitters and receptor proteins implicated in pain , which subsequently propagate pain signals to the central nervous system (CNS).2 Fasinumab is another agent in the final stages of trials for hip and knee OA. Fulranumab is a third agent that is no longer undergoing clinical investigation.It has been theorized that anti-NGF monoclonal antibodies might be able to intervene early in histamine activation and affect pain at the transcriptional level by decreasing production of cellular and subcellular elements critical to this pathway. Three antibody agents have been studied thus far. Tanezumab, a human immunoglobulin-2 NGF antibody, is being studied for osteoarthritis (OA), chronic low back pain, and metastatic bone lesions, among others. It has been found to be highly specific and selective for NGF, and in 2017 it was given fast-track status by the U.S. Food and Drug Administration. Meta-analysis of Phase 3 studies showed favorable outcomes versus placebo, naproxen, and oxycodone.2 Subsequently, studies for tanezumab were limited to lower doses. Other common side effects include arthalgias, nasopharyngitis, and paresthesias.Unfortunately, there may be significant limitations when using these current agents. In 2010 all trials of these agents were put on hold due to concerns of rapidly progressive osteoarthritis. Trials were resumed in 2015, with the exception of fulranumab. The meta-analysis for tanezumab has revealed that doses of 10 mg or higher increase the risk of rapidly progressive osteoarthritis (as high as 6.3%), particularly with concomitant nonsteroidal anti-inflammatory use and a history of fracture.2 Further trials ranging from 16 to 56 weeks of therapy demonstrated similar results with less success in the 2.5 mg dose range. With mindful patient selection these agents may be indicated as an alternate line of therapy for those who have failed conventional regimes.Despite adverse effects, trials for tanezumab demonstrated analgesic benefit in patients with OA and low back pain if limited to 2.5 mg or 5 mg doses for up to 8 weeks.In summary, though there is promising initial data to support NGF antagonists as an analgesic modality, the current agents studied generally have side effects that outweigh the benefits, particularly at higher doses. It is hoped that dosing studies and further trials will elucidate a more favorable profile.Microglia are cells of the reticuloendothelial system of the CNS. Arising from myeloid precursors derived from embryonic mesoderm, these cells migrate to the CNS and become microglia. They are responsible for shaping and reshaping neuronal circuits continuously throughout an entire life span. They comprise 10% to 20% of all CNS cells, with the highest densities in the hippocampus, basal ganglia, substantia gelatinosa, and spinal cord. They are responsible for a significant portion of what we call neuroplasticity and are involved in the cellular processes that take place during and after nerve injury, one of the pathognomonic features of neuropathic pain.3–65When a nerve is injured, microglia undergo “microgliosis,” or activation and migration, which allows them to perform several responses, including protecting injured nerves, phagocytosis of cellular debris, and the release of cytokines and other inflammatory modulators to initiate and propagate an immune response. Persistent pain models have demonstrated that hyperalgesia is associated with microglial activation in the dorsal horn of the spinal cord.4 These systems affect intracellular transcription and play a critical role in inflammation. MAPK includes several signaling chemokines and cytokines, including extracellular regulated kinases, p38 protein, and c-Jun N-terminal kinase.4Microglial activation occurs through several pathways, perhaps most important the mitogen-activated protein kinase (MAPK) pathways.4 These cytokines are released by the damaged afferent neurons themselves to activate microglial p38 pathways and ultimately induce transcription of further inflammatory mediators, including cyclooxygenase-2, brain-derived neurotropic factor, IL-6, TNF-α, and IL-1β. These mediators then activate surrounding astrocytes, increase recruitment of more microglia, and promote CNS sensitization and neuronal transmission. Glial TNF-α also causes increased synaptic efficiency for propagation of pain signals, increases endocytosis of gamma aminobutyric acid-A receptors, and increases N-methyl-d-aspartate receptor receptor activity through extracellular regulated kinase phosphorylation.4 This is effectively a downregulation of the inhibitory pain modulation response, resulting in increased pain signaling to thermal and mechanical nociception and hyperalgesia.Microglial p38 MAPK pathways are activated via phosphorylation by many proinflammatory cytokines and mediators, including tumor necrosis factor-α (TNF-α), interleukins (such as IL-1β), and adenosine triphosphate (ATP).5 Studies so far are limited to animal models, and few look at adverse effects. It is theorized that intrathecal administration would minimize systemic effects of minocycline, and one study did not find any evidence of neurotoxicity.7 Differential target multiplexed (DTM) spinal cord stimulation (SCS) has been studied in the setting of targeting spinal anatomy known to be richer in glial cells, particularly the T8–T11 region. Ongoing human studies targeting these regions with DTM SCS have produced preliminary results showing improved analgesic outcomes over conventional SCS for back and leg pain.8 It is thought that SCS affects glial cell interactions, which include intra- and intercellular communication, signal transduction, modified protein phosphorylation, and ion transport.9Regarding therapeutic potential, intrathecal administration of p38 inhibitors such as minocycline, a clinically used antibiotic, inhibits microglial activation and decreases pain behavior by attenuating neuropathic pain.Microglial influence on neuronal plasticity and the ability to modulate this response may have large implications in the treatment, prevention, and management of chronic pain syndromes. A better understanding and development of pharmaceutical and neuromodulatory interventions for these pathways may allow physicians to reduce the burden of chronic pain after nerve injury, including postoperative pain syndromes.10 When a cellular stressor such as hypoxia, hypoglycemia, or chemical insult affects a cell, ATP levels decrease, which activates AMPK to restore the balance. AMPK also acts as an inhibitory regulator of several enzymatic pathways linked to chronic pain, including MAPK and mammalian target of rapamycin complex 1.11AMPK is an enzyme involved in cellular homeostasis that maintains cellular energy levels by regulating ATP production and consumption.11 Indirect activators enhance phosphorylation at multiple sites thereby increasing enzymatic activity. Two known AMPK activators include metformin and O304. Metformin, a clinically used anti-diabetic agent, acts as an indirect AMPK activator nonspecifically through several intracellular sites and can cross the blood–brain barrier. Conversely, O304 does not cross the blood–brain barrier and is a specific AMPK activator that decreases phosphorylation of the Thr172 site on the alpha subunit via protein phosphatase 2 C and does so without suppressing that enzyme’s AMPK activating effects.11Activation of AMPK can be achieved through several mechanisms. Direct allosteric activators protect the kinase from dephosphorylation via β or γ subunits.12 AMPK activators have also demonstrated complete resolution of long-standing nerve injury within 60 days of the event. One study looked at metformin and O304 in a postoperative pain mouse model and demonstrated reduced mechanical pain sensitivity with both agents compared with the drug vehicle. This study also demonstrated an even greater synergistic effect with both agents co-administered.12Mouse model studies have demonstrated that AMPK activation can reduce peripheral nerve injury and allodynia within 7 days of injury.11 These agents are 100 times more potent than metformin, activating AMPK by targeting specific γ-2 subunits for both strong activation and avoidance of adverse effects.10 As specific allosteric agents, they also have a diminished “scatter” effect compared with nonspecific activators. These types of agents may represent a more robust therapeutic pathway compared with the currently studied drugs.There are rich opportunities for further study and expansion within this field. Metformin studies may be designed to evaluate treatment of postsurgical and neuropathic pain, possibly as part of a preemptive perioperative regimen such as an enhanced recovery after surgery protocol. There is also a possibility for further development and study of other allosteric AMPK activators such as A769662 and OSU-53 for clinical use.Genetic studies have revealed that nociception, chronic pain syndromes, and analgesic management all have links to heritable traits. Knowledge in this area is still expanding. This section will summarize a variety of genetic issues in pain management, including enzymatic activity, addiction, behavior, mutations, channelopathies, and specific heritable pain disorders.13Most currently available genetic testing emphasizes drug metabolism and pain response. One well-described system involves opioid metabolism by the cytochrome P450 family of isozymes. There are quantitative and qualitative interpersonal variations of this isozyme cohort, which can cause very different clinical outcomes during opioid therapy.14 Roughly 7% to 10% of Caucasians, 1% of Asians, and 1.4% of African Americans are have a CYP2D6 deficiency rendering the drug ineffective.14 Some patients, including up to 10% of African Americans, have a genotypic variant for ultra-rapid metabolism15 that increases the rate of conversion to morphine, resulting in supratherapeutic drug levels that have adverse effects, including death. This heritable variability in enzymatic activity can clearly have very serious ramifications directly translatable to clinical practice and has led to an avoidance of these agents in pediatric populations after a number of reported drug-related fatalities.16A classic example is how CYP2D6 metabolizes codeine, an analgesically inactive prodrug, into morphine via O-demethylation.18 The presence of the G allele resulted in high dose requirements and higher pain scores than AA genotypes.18 High frequencies of the A118G G allele variants have been found in opioid-dependent subjects of both Hispanic17 and Swedish19 origin.Other examples include μ-opioid receptor gene OPRM1. This receptor has a known polymorphism identified in cancer patients named A118 G with three genotypes . Patients undergoing oxaliplatin chemotherapy with tramadol had differing analgesic outcomes based on allele genotype. The G allele is responsible for an amino acid change from asparagine to aspartic acid in the N-terminal of the μ-opioid receptor that may alter binding and/or response to substrates.20 The dopamine receptor has been a highly active target of study. Dopamine receptor D2 (DRD2) polymorphism is associated with addictive behavior; DRD2 gene Taql RLFP A (rs1800497) was studied in patients with an opioid use disorder versus control. Patients with either A1A1 or A1A2 alleles consumed twice as much heroin as those without the A1 allele.21 This allele is present in 19% of Caucasians with opioid dependence compared to 4.6% of those without drug abuse history.22 Dopamine D3 receptors are prevalent in the reward and reinforcement center of the nucleus accumbens. Opioid-dependent patients with high sensation-seeking scores are more likely to be homozygous for the DRD3 allele compared to patients with lower scores.23Addictive behavior has also been identified as having a strong genetic association, with 43% of the variance in drug abuse due to genetic factors unique to opioid metabolism.− channel), long QT syndrome types 1 and 2 (delayed K+ channel), long QT syndrome type 3 (Na+ channel), epilepsy (voltage-gated Ca++ channel), and diabetes mellitus (ATP-sensitive K+ channel).24There are other non-opioid-related genetic pathways implicated in acute and chronic pain syndromes. One area of developing knowledge involves channelopathies. This term refers to a loose organization of more than 30 heterogeneous conditions that all share the root cause of genetic defects in ion channel function. These defects may be inherited  or acquired . Commonly recognized non-pain examples include cystic fibrosis  in which a single base-pair substitution occurs in the genetic code at a specific point in the genome. This base pair can then have a downstream effect on the gene expression, including qualitative and quantitative defects of the resulting protein complexes. SNPs that occur together with high frequency are often referred to as a given haplotype. Haplotypes help identify other polymorphic sites on the same chromosome and are implicated in the pathogenesis of certain genetically linked disorders. There are more than 400 genes that encode human ion channels, and an almost endless number of permutations of mutations can occur.25 Catecholamine-O-methyltransferase is responsible for breaking down adrenergic and dopaminergic substances; SNPs and haplotypes of decreased catecholamine-O-methyltransferase activity are associated with increased pain sensitivity.26 Guanosine triphosphate cyclohydrolase haplotypes are similarly associated with decreased pain in both cancer-related and benign (laminectomy-related) pain.27 Pain catastrophizing behavior characterized by high levels of anxiety, vulnerability, and negativism has been linked to the presence of a short allele in the promoter region of 5-HTTLPR, a serotonin transport gene.28SNPs in various genes have been shown to have relevance for pain sensation, either decreasing or increasing pain perception. Various ion channels are involved in many aspects of in pain signal transduction, transmission, and modulation. Haplotypes of the ion channel genes SCN9A (sodium channel), CACNA2D3 , KCNS1 (potassium channel), CACNG2 , and P2RX7 (ATP-gated ionotropic receptor) have been associated with altered pain sensation postoperatively.v1.7 (encoded by SCN9A) is implicated in painful and painless conditions.25 This sodium channel subtype is expressed selectively in sensory and autonomic neurons. Mutations that inactivate the SCN9A gene result in both congenital insensitivity to pain and hereditary sensory and autonomic neuropathy type IID.25 Gain-of-function mutations in this gene produce syndromes such as inherited erythromelalgia, paroxysmal extreme pain disorder , and small-fiber neuropathy.25 Other voltage-gated sodium channel subtypes have also been associated with pain disorders, including Nav1.8 (SCN10A) with small-fiber neuropathy and Nav1.9 (SCN11A) with congenital insensitivity to pain.25The voltage-gated sodium ion channel NaTo summarize, there are strong implications regarding a patient’s pain experience and analgesic outcomes as directed by his or her genetic code. Enzymatic activity can significantly alter sensation, addictive behaviors, and medication metabolism, complicating a clinical picture. SNPs and other mutations in ion channel genes may lead to channelopathies that may fundamentally alter how a patient experiences and responds to a nociceptive stimulus. Moreover, some patients may suffer from painful conditions entirely caused by a genetic mutation resulting in a defective channel protein. Understanding of opioid metabolism variability has influenced prescription practices, most notably codeine use in pediatric populations. There is little use of genetic testing in practice today; looking to the future, genetic mapping and screening may help provide direction for pharmaceutical management and abuse risk. Gene therapies may represent a means to affect and interact with these complex systems to prevent or treat acute and chronic pain.This review has introduced four novel areas of pain research that show promise in developing both a better understanding of pain responses and new target-specific analgesics for persistent pain.Targeting of persistent pain mechanisms is focused on preventing or altering neural plasticity. Specific receptor or transmitter agents are acting on key steps in the pain cascade. Personalized medicine through genotyping will reduce risk and improve outcomes using genetically compatible opioid targets.The cascade of pain transmission is becoming ever clearer yet incredibly complex. There are limitations to the current clinical impact of these areas of study; prospective new agents are either not U.S. Food and Drug Administration approved or are not readily available for immediate general use. NGF antagonists require in-depth assessment of risk–benefit ratios before using in patients with osteoarthritis.


There are approximately 1.2 million people currently living with spinal cord injury (SCI), with a majority of cases at the cervical level and half involving incomplete injuries. Yet, as most preclinical research has been focused on bilateral thoracic models, there remains a disconnect between bench and bedside that limits translational success. Here, we profile a clinically relevant model of unilateral cervical contusion injury in the mouse . We demonstrate sustained behavioral deficits in performance on grip strength, cylinder reaching, horizontal ladderbeam and CatWalk automated gait analysis tasks. Beyond highlighting reliable parameters for injury assessment, we also explored the effect of mouse strain and age on injury outcome, including evaluation of constitutively immunodeficient mice relevant for neurotransplantation and cellular therapy testing. Comparison of C57Bl/6 and immunodeficient Rag2gamma(c)-/- as well as Agouti SCIDxRag2Gamma(c)-/- hybrid mouse strains revealed fine differences in post-injury ipsilateral grip strength as well as total number of rearings on the cylinder task. Differences in post-SCI contralateral forepaw duty cycle and regularity index as measured by CatWalk gait analysis between the two immunodeficient strains were also observed. Further, assessment of young (3–4 months old) and aging (16–17 months old) Rag2gamma(c)-/- mice identified age-related pre-injury differences in strength and rearing that were largely masked following cervical contusion injury; observations that may help interpret previous results in aged rodents as well as human clinical trials. Collectively, the work provides useful insight for experimental design and analysis of future pre-clinical studies in a translational unilateral cervical contusion injury model. As many as 1.2 million people are currently living with spinal cord injury (SCI), with a majority of cases involving the cervical level. Further, about half of all SCI cases involve incomplete injuries, in which the clinical effects are unilateral or do not affect both sides of the body equally [Given the anatomical and physiological differences between the cervical and thoracic spinal cord, there is growing evidence of unique pathophysiology that may affect the efficacy of therapeutic strategies . SpecifiThere have been previous descriptions of unilateral cervical contusion SCI in rodents –8, demonAll in vivo experiments were performed with the approval of the Institutional Animal Care and Use Committee at the University of California, Irvine. The combined experimental overview is shown in 8-week old female C57Bl/6 mice were divided into five injury groups (N = 12 per group) of varying severity, in which an IH Impactor (Precision Systems and Instrumentation) was used with a force of 30kD. Injury severity was increased by varying the dwell time within the IH Impactor settings  to create injuries of varying severity. It is important to note that the dwell time parameter is in addition to the spinal cord contact time, which reflects the time it takes for impactor to reverse direction. The contact time for the 0s dwell time is 4.45ms ± 0.53ms. The uninjured control group received a laminectomy alone. Under 2% Isoflurane anesthesia, mice received a laminectomy at the C5 vertebral level and the lateral vertebral processes of C4 and C6 were cleared of muscle to allow for stabilization of the vertebral column using 0.8mm tip forceps attached to the clamping platform. The impactor tip (1.0mm diameter) was consistently positioned on the right side of the spinal cord, between the midline of the spinal cord, and the lateral edge of the C5 vertebrae, to create a unilateral injury. The muscle was sutured with 5–0 chromic gut, and skin closed with 7 mm wound clips. After surgery, mice were allowed to recover overnight in cages with Alpha-Dri bedding (Newco Distributors Inc.) placed on water-jacketed heating pads at 37°C. Post-op care consisted of lactated ringers (50 ml/kg s.c.) once a day for 5 days, Baytril  for 2 weeks, Buprenorphine (0.01mg/kg s.c.) immediately after injury and every 12 hours for 2 days thereafter as well as manual bladder expression twice a day until mice recovered some bladder expression, then once a day for the duration of the study. All surgeries were done by personnel previously trained to perform consistent laminectomies and injuries, as validated by IH device feedback and monitoring of behavioral outcome.For mouse strain evaluation, the 8-week old female C57Bl/6 were compared with Rag2gamma(c)-/- (Taconic) as well as Agouti SCIDxRag2Gamma hybrid (StemCells Inc) mice using a C5 vertebral unilateral contusion with a force of 30kd and 5s dwell time (N = 10–12 per group). The same parameters were used to compare the effects of age, where young (15–17 weeks) or aged (65–68 weeks) Rag2gamma(c)-/- mice underwent the aforementioned C5 vertebral unilateral contusion with a force of 30kd and 5s dwell time (N = 12–14 per group). Due to the immunodeficient nature of the Rag2gamma(c)-/- strain, additional antibiotics were administered daily throughout the 13-week duration of the study .All in vivo work included random allocation into study groups. All experimental data collection and analysis was completed by individuals blinded to the study groups. Further, all neurobehavioral tasks were preformed pre-injury and all animals were pre-trained on ladderbeam, CatWalk and grip strength tasks to prevent exploratory behavior.Grip strength of each forepaw was measured independently using a Dunnett-style grip strength meter . BrieflyLeft  and right  forelimb asymmetry was assessed using the cylinder task. Mice were placed in a glass beaker for 5 minutes, with 2 trained observers on opposite sides. The number of placements of either or both paws on the sides of the beaker were tallied and results were reported as percent of total placements for the right  paw or for both paws. The total number of rearings during the testing period was also compared.Animals were evaluated for stepping errors when walking across a horizontal ladder with 50 rungs analyzed . The tasThe Catwalk XT 9.1 gait analysis system (Noldus) is used to analyze a wide variety of gait parameters in rodents . We analAt 8 weeks  and 13 weeks (age-comparison) post-injury, mice were injected with a lethal dose of pentobarbitol followed by cardiac perfusion with 1% PBS, followed by 4% paraformaldehyde (PFA) in 1% PBS (pH 7.4) and the spinal cord dissected as previously described . Spinal An Olympus BX51 microscope with motorized stage and StereoInvestigator were used for unbiased estimation of total cell numbers  and lesion volume . Uniform random sampling of the tissue was performed according to standard stereological principles, where sampling parameters (i.e. grid size and counting frame size) were empirically determined to arrive at low coefficients of error (CE). CE is defined as the standard error of the mean of repeated estimates divided by the mean and used as a measure of the accuracy of the stereological procedure. The probe grid size and counting frame size were empirically determined to yield average cumulative error values <0.1. For the Cavalieri probe, the section evaluation interval was 6 with a distance of 180μm between sections. Cell counts were performed with a counting frame of 50x50μm.Statistics were performed using GraphPad Prism (version 6.0). Comparison of behavioral results between groups at a single timepoint, histological lesion comparisons were analyzed for statistical significance, using one-way ANOVA, with Tukey multiple comparisons post-hoc, or t-tests where appropriate. Repeated measured ANOVA was applied for behavioral data over time, while rostrocaudal evaluation of histological data was analyzed for statistical significance using two-way ANOVA, with Tukey multiple comparisons post-hoc. The results for behavioral parameters Correlation results between histology and behavioral tasks were evaluated with parametric Pearson analysis .Principal component Analysis (PCA) was performed using the “prcomp” function in R via RStudio. PCA is a feature extraction tool, in which an orthogonal transformation is applied to a set of input variables in order to identify a set of factors, termed principal components, that account for the variability in the dataset. Generally, only the first 1–3 PCs are needed to explain the majority of the variance, allowing the other PCs to be discarded. Further, variables that correlate will have high “loadings” (contributions) to the same PC. In this way, a data set with a large number of variables can be reduced to a smaller number of variables. Retention of PCs was determined by the “Kaiser Rule” , in which a PC is found to account for more variance than a single variable from the original dataset. Retained PCs were used to score each injury group, the resulting mean scores plotted, and one-way ANOVA with post-Hoc HSD tests were used to test for significant differences between individual groups.Animal exclusions were determined according to pre-hoc criteria by an investigator that was blinded to the experimental groups. In the C57Bl6 mouse cervical injury profile, N = 12 per group was planned for the study, with the exception of the most severe injury group (10s dwell) which had N = 14. Two animals were excluded from the all final data due to surgical complications . Two additional animals were excluded from catwalk analyses as data outliers (Grubbs Test). In comparison of strain (C57Bl/6 with Rag2gamma(c)-/- mice) and age, a total of 2 animals died during the course of the study with no identified cause other than age-related mortality.C57Bl/6 mice underwent a right-sided unilateral contusion (30kd force) at the C5 vertebral level with varying the dwell time  of the impactor probe on the exposed spinal cord. Injury effects in grip strength, cylinder task, horizontal ladderbeam and CatWalk were sustained at the 8-week assessment .Ipsilateral grip strength testing revealed a sustained impairment , in whicThe cylinder paw placement task revealed a severe impairment of the ipsilateral forepaw , in whicStepping errors on the horizontal ladderbeam task also showed a persistent injury in ipsilateral forelimb function , with siOverall changes in gait were assessed via CatWalk parameters , where sLastly, it is also important to note that behavioral deficits were likely unaffected by either thermal hypersensitivity or mechanical allodynia, as there were no significant differences were observed in ipsilateral or contralateral Hargreaves latency . SimilarIn general, increasing the dwell time of the impactor probe led to greater injury severity as measured by ipsilateral grip strength, bilateral placements on the cylinder task, ipsilateral horizontal ladderbeam errors and CatWalk gait parameters. Importantly, most neurobehavioral tasks were unable to discriminate function between uninjured and 0s dwell time at the 8-week timepoint post-injury, apart from % Aa stepping pattern during locomotion. Further, neither task was able to identify differences between 2s and 10s dwell time, suggesting a limit in detection sensitivity. While we only report several key parameters, there are others that may highlight improvements following therapeutic intervention and should be reviewed on a case-by-case basis.Histological analysis was performed at 8 weeks following injury to evaluate the effect of dwell time on neuroanatomical integrity. White matter sparing (MBP) and neuronal loss (NeuN) were assessed via stereological analysis of sections rostral and caudal to the injury epicenter . ChangesThere was minimal neuronal loss contralateral to the injury, with a significant difference detected only between the 5s dwell time and the uninjured cord 0.36mm from the epicenter . There wTaken together, white matter (MBP) damage, neuron (NeuN) loss and astrogliosis (GFAP) were detected predominantly ipsilateral to the cervical contusion. As with behavioral data, most differences between injured groups with varying dwell times did not reach statistical significance. Only neuron counts rostral and caudal to the injury epicenter were sensitive enough to distinguish between the injured groups with varying dwell times and the injured group without a dwell time. Above all, the reported changes in neuroanatomical integrity demonstrate that there is a relationship with functional deficits; we explored this relationship further via correlation analysis.With few detectable differences between injured groups of varying dwell times, correlation analysis between function and neuroanatomical integrity was predicted to reveal more subtle changes. Specifically, we compared locomotor function  with measured histological parameters . ImportaAlthough there was only a weak correlation with number of neurons on the ipsilateral side and Catwalk forelimb max area , there was also a correlation noted with the contralateral side . This was the only significant correlation between function and contralateral histology, and could be indicative of contralateral plasticity that is involved in forelimb paw placement during gait. However, as plasticity was not evaluated in this study, this should be properly addressed in future work. The number of ipsilateral NeuN-positive cells also correlated with ipsilateral ladderbeam errors , as well as percentage of both paw placements on the cylinder task .Specific to the ipsilateral spinal cord, GFAP as well as lesion volume displayed only a weak correlation to ipsilateral ladderbeam errors , percentage of both paw placements on the cylinder task  and grip strength . In summary, the strongest correlations with function were obtained from histological assessment of ipsilateral white matter sparing (MBP) and neuron (NeuN) counts. Together, these data highlight a linear relationship between behavioral outcomes and neuroanatomical integrity using the dwell time (IH impactor setting) to create injuries with varying severity.−10) accounted for ~ 72% of the variance in the dataset  was employed to further investigate the behavioral outcomes observed to be strongly correlated with histology . Analysi dataset .−6) was similarly affected by GFAP area, lesion as well as fibronectin volume, and ladderbeam forepaw errors. In the opposite direction, PC1 was driven by right forepaw grip strength (Grip RF), percentage of bilateral placements on the cylinder task and CatWalk maximum forepaw contact area, accounting for ~ 63% of the variance in the dataset  unilateral cervical contusion were similar between C57BL/6, Rag2gamma(c)-/- (Rag2g (c)) and SCIDxRag2Gamma hybrid (Rag2g (c) hybrid) mice at 8 weeks post-SCI . NeverthAs age is thought to play a role in pathogenesis post-SCI, functional deficits and histological parameters were compared between young (3–4 months) and aging (16–17 months) Rag2Gamma mice post-SCI. In uninjured animals, there were significant  age-related differences in contralateral and ipsilateral grip strength . InteresThere was also a minor decrease in the percentage of placements of both paws during the cylinder task in aging mice following injury , but thiIn support of the modest behavioral differences between young and aging mice, there were no effects  in contralateral and ipsilateral white matter sparing  or neuroIn this study, we describe sustained behavioral deficit and tissue pathology following a unilateral cervical contusion spinal cord injury (SCI) in mice. Beyond highlighting reliable parameters for injury assessment via a varying dwell time paradigm, the work also explores mouse strain and age effects on injury outcome. The comparison between C57BL/6, Rag2gamma(c)-/- (Rag2g (c)) and SCIDxRag2Gamma(c)-/- hybrid (Rag2g (c) hybrid) mice strains provides evidence of fine differences in deficit post-injury, specifically in ipsilateral grip strength and total number of rearings on the cylinder task. There were also differences in forepaw duty cycle and regularity index between Rag2g (c) and Rag2g (c) hybrid mice at 13 weeks post-SCI. Altogether, this study provides useful insight for experimental design and analysis of future pre-clinical studies in two translational SCI models.As a most clinical spinal cord injuries are incomplete and at the cervical level, a reliable research model to serve as a basis for basic research studies and testing therapeutics is critical for progress in the field . FurtherInterestingly, unlike the previous unilateral cervical model in mice , our injDespite several reports of mouse strain differences in functional recovery and tissue pathology following thoracic SCI –13, the Similarly, young and aged mice were compared in their response to the unilateral cervical contusion injury. Previous work with thoracic models of SCI has shown aged rodents have less locomotor recovery, a larger area of pathology and more axonal demyelination compared to younger rats . FurtherCollectively, this work provided a thorough characterization of the unilateral cervical contusion injury, with sustained functional deficits detectable on multiple behavioral tasks. Although the goal was to develop a gradient of neuroanatomical and functional deficits, only ipsilateral ladderbeam error values were sensitive enough to detect the differences between different dwell times. Further, although it increasingly recognized that a single outcome is unlikely to capture the complexity of impairment post-SCI  and multS1 Fign = 12, 11, 11, 10, 14 for U, 0, 2, 5, 10 groups). Data are expressed as mean ± SEM .Contralateral and ipsilateral results are shown for all relevant parameters ((TIF)Click here for additional data file.S2 FigData are expressed as mean ± SEM  with r and p-values reported.(TIF)Click here for additional data file.S1 Method(DOCX)Click here for additional data file.


Monochorionic twinning involves numerous maternal and fetal complications, triplets complicated by a monochorionic pair are at further increased risk. Here, we report a case of repeated triplets complicated by monochorionic diamniotic twins with successful pregnancy outcomes in a woman using autologous oocytes.A 30-year-old female undergoing embryo transfer with fresh and frozen embryo cycles with autologous oocytes. The two cycles were confirmed by transvaginal ultrasound to result in successful clinical pregnancies of triplets complicated by a monochorionic twinning. The first pregnancy resulted in a singleton delivery after a selective reduction of the monochorionic pair. The subsequent pregnancy resulted in a dichorionic diamniotic twin pregnancy after the heartbeat of one of the monochorionic twin fetuses stopped at 43 days after embryo transfer. Both of the pregnancies ended with successful live births.Our case report of repeated triplets with monochorionic twins suggests the potential causes and risk factors of monochorionic twinning in assisted reproduction and raises concern regarding the timing of multifetal pregnancy reduction. Many studies have demonstrated that an increased risk of monozygotic twins(MZT) was associated with assisted reproductive techniques(ART) procedures –4. AccorA couple who complained of secondary infertility for 6 years underwent IVF treatment due to polycystic ovarian syndrome and male ejaculatory dysfunction. The patient was a 30-year-old female, her cycle was followed with a standard long protocol for 18 days that included uFSH  and HMG . Final oocyte maturation was triggered by the administration of 10,000 IU of HCG . Oocyte retrieval was carried out by the transvaginal ultrasound-guided puncture of follicles. Sixteen mature oocytes were retrieved under transvaginal ultrasound guidance, of which 13 were fertilized after intracytoplasmic sperm injection(ICSI). Finally, 7 good-quality embryos were retrieved. Embryo culture in vitro showed two 8-cell embryos (grade A) and two 9-cell embryos (grade B) on day 3. Other embryos were culture to day 5 and yielded 3 viable blastocysts.Two cleavage embryos (one 8-cell embryo and one 9-cell embryo) were transferred on day 3 in the fresh cycle. The other two day 3 embryos were cryopreserved. Luteal progesterone support was given after the oocyte operation. The patient had confirmed pregnancy by a urine pregnancy test on day 14 post-transfer. Dichorionic triplets were diagnosed by a transvaginal ultrasound scan at 5 weeks post-transfer, as two gestational sacs (GSs) inside the uterus and two pulsating echoes were detected in one of the GS  was used in the first frozen cycle. A single blastocyst (Gardner’s classification: 4AA) was transferred, and the transfer failed. During the next cycle, two day 6 embryos (Gardner’s classification: 4CB*2) was transferred, but the transfer failed again. The woman successfully conceived in the following cycle after two cleavage embryos (8 cells grade 2 and 9 cells grade 2) were transferred. We first suspected dizygotic twins, as two gestational sacs (GSs) was detected with transvaginal US on day 30 post-transfer. However, the US showed two GSs with three fetuses one week later, and another dichorionic triamniotic triplet pregnancy was suspected Fig. B. The heThe data showed that there may be a significantly elevated risk of monozygotic twins after ART compared to natural conception , 3. MonoIt is plausible that an artificial breach in the ZP caused by the ICSI, assisted hatching(AH) or preimplantation genetic diagnosis may cause splitting of the inner cell mass(ICM) and MZ twinning , 16. HowRecent studies reported that the transfer of a blastocyst resulted in a higher incidence of MZ pregnancies than the transfer of cleavage-stage embryos –22. Our It has been shown that multiple pregnancies are associated with an increased incidence of abortion and premature labor. Triplets complicated by a monochorionic twin are at further increased risk of many maternal and fetal complications . A selecTo our knowledge, this is the first reported case of repeated triplets complicated by a monochorionic pair in a patient with autologous oocytes following assisted reproduction treatment. Potential risk factors for this patient include young maternal age, high quality embryos and micromanipulation of the ZP. In the cases with dichorionic triplets, selective multifetal pregnancy reduction is associated with a better obstetric outcome and should be offered in the obstetric management. While these cases are no longer rare after an ART procedure, the potential causes and risk factors of monochorionic twinning in assisted reproduction and the timing of MFPR warrant continued investigation.