Parameters of involved muscles showed no different from those of NAEOMs excluding 25th, 50th percentiles, and standard deviation (SD) (p < 0.05). NAEOMs displayed higher values of 90th, 95th percentiles, SD, skewness, inhomogeneity, and entropy than HCs (p < 0.05). ROC curve analysis of entropy yielded the best area under the ROC curve  for differentiating NAEOMs and HCs. After treatment, histogram parameters including 5th, 75th, 90th, and 95th percentiles, SD, kurtosis, inhomogeneity, and entropy were reduced in NAEOMs (p < 0.05). T2 histogram analysis could detect early involvement of EOMs in TAO prior to detection on conventional orbital MRI.Using histogram analysis of T2 values to detect early involvement of extraocular muscles (EOMs) in patients with thyroid-associated ophthalmopathy (TAO). Five EOMs of each orbit were analyzed for 45 TAO patients and 22 healthy controls (HCs). Patients’ EOMs were grouped into involved or normal-appearing EOMs (NAEOMs). Histogram parameters and signal intensity ratios (SIRs) of EOMs were compared; receiver operating characteristic (ROC) curve analysis was performed to differentiate NAEOMs from EOMs of HCs. 24 patients were reassessed following immunosuppressive treatment. For SIRs, involved muscles showed higher values than those of NAEOMs and HCs ( The current evaluation system is primarily based on the anterior visible part of the orbit and ignores deep structures, such as the extraocular muscle (EOM)2. EOM is one of the target organs of immune attack in TAO, its enlargement can produce severe complications3. As early as 19794, researchers found that approximately 70% of patients with Graves’ disease, who commonly characterized by subclinical ophthalmopathy, can present with EOM enlargement on computed tomography (CT) or magnetic resonance imaging (MRI). Therefore, the involvement of EOM may begin much earlier than is currently clinically recognized; the ophthalmic signs or symptoms-based evaluation system may be inaccurate, thus, lead to untimely and inappropriate treatment.Thyroid-associated ophthalmopathy (TAO) is an autoimmune disease characterized by orbital tissue inflammation, expansion, fatty infiltration and fibrosis5. However, most of the MRI methods are either semi-quantitative or based on signal intensity, which are not typically directly proportional to specific tissue properties. Additionally, these techniques mainly quantify disease activity, severity or therapy response; there is minimal attention to detection of the early involvement of EOMs. Politi et al.2 and Kilicarslan et al.6 suggested that diffusion-weighted imaging (DWI) could detect early asymptomatic involvement of EOMs prior to abnormality on conventional orbital MRI. However, DWI exhibits some inherent limitations in orbital imaging, particularly in terms of susceptibility to artifacts and signal loss7.Multimodality orbital , for its excellent soft tissue contrast, is viewed as an objective and sensitive tool to assess the EOMs from anatomy to tissue components in TAO8 and effectively characterize subtle changes from disease process or therapy before they manifest as signal abnormalities10 However, T2 values are frequently obtained from manually traced regions of interest (ROIs) on a single slice11, rather than integral muscle, resulting in measurement sampling errors and subjective bias. Recently, application of T2 for TAO have been reported10, but they also focused on staging and activity and have not yet been used to identify early involvement of EOM, and the information typically provided by mean T2 is rather limited. Volumetric histogram analysis of T2, measure the variations and frequencies of T2 values within whole tissues, can provide comprehensive and informative T2 parameters, is expected to potentially detect early EOM involvement even when there are no changes on conventional images.T2 mapping was considered as a tissue-specific technique can reflect biochemical components within EOMsBased on previous reports and on our own clinical observations, we developed the hypothesis that all EOMs of TAO patients may experience immune damage during early disease stage. Therefore, we aimed to detect this early involvement using volumetric T2 histogram analysis.In this study, 383/450 EOMs of untreated TAO patients were designated as the “involved muscle group” (n = 383) and 67/450 were designated as the “normal-appearing muscle group (NAEOMs)” (n = 67) by assessment of muscle involvement on conventional T2WI images. In addition, 220 EOMs of healthy controls (HCs) were included. In total, 191/240 involved EOMs and 49/240 NAEOMs of 24 patients who received treatment were also compared following therapy.Overall, the inter-observer agreement between the two readers was good to excellent for SIRs and volumetric T2RT histogram parameters (Table p < 0.001), and between involved and NAEOMs (p = 0.04). There was no statistical difference between NAEOMs and HCs (p = 0.21) . Heterogeneous metrics, in addition to the 5th and higher  percentiles of T2 of NAEOMs showed marked reductions after therapy  elevated percentiles and increased heterogeneous distribution of T2 can occur in all EOMs of TAO patients, including involved or NAEOMs on conventional MRI; (2) the NAEOMs exhibited similar changes in percentiles and non-uniform T2 distribution in comparison to involved muscles; (3) NAEOMs showed much higher values in 90th and 95th percentiles T2 than those of HCs.13 and pelvic limb muscles14. Ishu et al.9, demonstrated that the lower leg muscles of boys with Duchenne muscular dystrophy exhibited elevated percentiles and heterogeneities on T2-mapping histogram analysis. Inflammation, fatty degeneration and fibrosis were main pathological changes of EOMs in patients with TAO, there is no absolute boundary between the fatty infiltration and edematous, and both of them can lead to increased T2 values15. Heterogeneous expression at the cellular and molecular levels, including fibroblast phenotypes, cytokine profiles, and disparate T cell subsets16, may produce the asynchronous and non-uniform pathological changes in each EOM within the same orbit, thus the microstructural component distribution within each EOM may be uneven. The varying deposition of macromolecules , interstitial edema, and fibrous scar tissue and fatty infiltration within the involved muscles can result in greater distribution of T2 values18. Factually, uneven distribution of pathological components in EOMs in patients with TAO has been reported by Yokoyama et al.19 and it was observed to have correlation with the therapeutic response to corticoid treatment. However, the uniformity in Yokoyama’s report was based on visual observation of T2 signal intensity of EOMs on conventional MRI; notably, T2 SI is variable and does not directly reflect tissue properties, such as density or molecular composition.We found that the involved EOMs exhibited overt increase in point-specific parameters (excluding the 5th percentile) and more inhomogeneous distribution of the T2 than EOMs of HCs. Histogram features provide novel quantifications of the intrinsic distributions of different tissues, such as percentiles providing a greater range of T2 values to reveal complex tissues; these have been widely used to quantify the T2 distribution in muscles such as myocardium6, who observed changes in diffusion values but without significant differences. We speculate the changes in NAEOMs are from the following factors: (1) previous T2 studies on EOMs in TAO primarily focused on altered T2, averaged over one or several image slices20, which may harbor or mask small pathological changes. These subtle pathologies could be diffusely or focally distributed throughout the EOM, such that they could not be captured by conventional MRI. Histogram-derived percentile parameters, in which the T2 values were arranged from small to large, may enable distinction of subtle differences. (2) Lower percentiles of T2 represent authentically normal muscle area, while the mean and higher percentiles  of T2 may represent abnormal voxels. In NAEOMs, large amounts of normal voxels masked tiny amounts of abnormality; consequently, only very high percentiles of T2 reflecting extremely abnormal voxels could detect this subtle modification. (3) Secondary to long-standing systemic immunoreactions, NAEOMs also experience lymphocyte infiltration and accumulation of granular material. These microstructural changes precede functional, morphological, and signal changes, which may be a hallmark of early muscle involvement and are difficult to discern by conventional means.Interestingly, the NAEOMs exhibited larger values in both inhomogeneity metrics and higher percentiles (90th and 95th) of T2 values, relative to those of HCs. However, no differences were observed in the SIR, mean, and lower percentiles of T2. Our findings regarding the early involvement are similar to, but more obvious than, those reported by Kilicarslan et al.Notably, overtly involved muscles displayed higher 25th and 50th percentiles, compared to those of NAEOMs. This is potentially because both 25th and 50th percentiles were located in borderline areas between minimal abnormality and disease. Most of the distribution shape-related scalars of NAEOMs (with the exception of SD and inhomogeneity) displayed no obvious differences from those of involved muscles. These results further supported the immunologic injury in NAEOMs.mean value had lower AUC when compared to the higher T2 percentiles and entropy. It demonstrated that conventional MRI features and commonly used T2 may have some limitations in detecting the early changes of EOMs in TAO. The entropy refers to irregularity of gray-level distribution, higher entropy reflects greater complexity of specific tissue21 and indicates the diversity tissue component. This finding suggest that the quantification of macromolecules can be used as a direct imaging marker of early involvement.In our present findings, the entropy had the best diagnostic performance among the T2 histogram parameters and SIRs. Both the SIRs on conventional MRI and the usually used T2In this study, in patients who displayed clinical amelioration, apart histogram parameters for involved EOMs changed. The 5th and higher  percentiles of T2 and all uniformity-related parameters of NAEOMs decreased markedly after therapy. Therapy-induced modifications of NAEOMs indirectly demonstrated that they exhibited some pathology.Our results are consistent with the immunological nature of TAO: when immune activity is triggered, all EOMs experience varying degrees of immune reactions. EOM damage begins much early prior to the onset of overt clinical signs and symptoms, as well as before detection on routine orbital MRI; thus, comprehensive and meticulous evaluations should be performed to detect such changes. This rationale explains the phenomenon we frequently encountered in clinical practice: many patients who initially manifested with mild involvement in some EOMs, which quickly progressed to severe ophthalmopathy and involved all EOMs. It can also partially explain why local therapy is less effective than systemic intravenous injection, even in TAO patients with isolated muscle involvement. Thus, our results may promote recognition of the traditional definition of “non-involvement” based on clinical assessment, gross morphology or signal intensity on conventional MRI. The histogram parameters of T2 may offer an data-based evidence to facilitate improved clinical treatment of TAO.This study had some limitations. First, it did not include a “gold standard,” such as histological or immunohistological analyses, which could have verified the microscopic alterations behind the image analysis. Notably, this verification may be conducted in appropriate animal models. Second, the neuroradiologists were aware of the subjects’ conditions when identifying muscle involvement on conventional T2WI images; they assumed that the EOMs of HCs were normal and did not assess their involvement. Third, the T2 mapping technique were performed without fat suppression, and we cannot really distinguish the altered value were from fat or edemas, but both of them were pathology changes in TAO. Fourth, the T2 values calculated in this research still failed to overcome the inherent drawbacks that the contamination from stimulated and indirect echoes, which thus give rise to deviated and inaccurate T2 values. Finally, the sample size of NAEOMs was limited, and patients were not classified into subgroup according to varying activity, which may have reduced the statistical power of the study.The combination of histogram analysis and T2-mapping imaging can detect early pathology in EOMs without obvious involvement on conventional orbital MRI. This multiparametric approach may provide a breakthrough for exploring early injuries in EOMs for patients with TAO. Early intervention may be started in patients with highly abnormal histogram parameters regardless of the CAS score and when there is no evidence of EOM involvement on conventional imaging. This information may aid in the management of TAO.This retrospective study was approved by the institutional review board of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. Written informed consent was obtained from all patients for MRI scanning, but the written informed consent of the data usage for this research was waived as the MRI protocol is conventional in our hospital and this is a retrospective study.22. Patients were enrolled if they met the following criteria: (1) initially diagnosed with active moderate-severe TAO; (2) showed no other accompanying orbital disease; (3) had no prior orbital therapy or surgery; (4) successfully completed MRI examinations.Between May 2017 and Apr 2018, TAO patients and healthy controls (HCs) were recruited. An endocrinologist and an ophthalmologist jointly diagnosed the TAO according to the consensus of the European Group on Graves' OrbitopathyFinally, 45 active moderate-severe TAO patients  met the inclusion criteria. In addition, 22 HCs  were included; they were euthyroid healthy volunteers without any ocular symptoms or signs of exophthalmos .24 patients who completed therapy and exhibited a favorable therapeutic response were further assessed. And all of them were received the standard therapy regimen which were recommended by 2016 EUGOGO guidelines: intravenous methylprednisolone (Pfizer Manufacturing Belgium NV) 0.5 g per week for 6 weeks, followed by 0.25 g per week for 6 weeks. The total therapy course lasted for 12 weeks.Orbital MRI was performed 1 week before the initial treatment for patients. Patients who underwent therapy were re-scanned within 2 weeks after the completion of therapy. All MRI scans were performed on a 3T MRI scanner  with a 32-channel head coil. Subjects were asked to keep their eyes closed during scanning, to facilitate the subjects to keep the eye closed, some auxiliary does not interfere with magnetic fields were covered on their eyes.The MRI protocol was as follows: (1) axial T2WI ; (2) coronal T2-IDEAL . (3) coronal T2-mapping with eight consecutive echoes  and the following parameters: TR, 2000 ms; section thickness of 3 mm, gap of 0.6 mm; FOV, 180 × 180 mm; matrix, 256 × 256; and bandwidth, 15.63 kHz/pix. Acquisition time for T2-mapping was approximately 6 min 24 s.The axial T2WI images were traditional orbital images to exclude other obvious ocular disease. The T2-IDEAL technique was utilized to observe morphological changes and assess SIRs. The coronal T2-mapping sequence was employed to assess T2 values.The following image analyses were performed by two experienced neuroradiologists with more than 7 years of experience. The neuroradiologists were blinded to the clinical data and subjects’ conditions (patients or HCs), although the images may have suggested some characteristics of the subjects.(1) Signal intensity measurements24, on a GE AW 4.6 workstation . The neuroradiologists manually outlined the ROIs of five EOMs  for each orbit. They delineated the muscles on the slice with the most prominent signal intensity alterations on water images of the coronal T2-IDEAL sequence. Because separation of the superior rectus and levator palpebrae superioris is difficult, these muscles were sketched together as the superior muscle group  Volumetric T2 histogram analysisMRI data for the T2 maps were digitally transferred to a personal computer and processed with custom-developed software . Post-processing was conducted using the monoexponential T2-mapping fitting model.All ROIs of EOMs were traced by the two neuroradiologists. The ROIs were manually drawn on all contiguous slices  along the edge of the EOMs to encompass the whole muscle. The neuroradiologists were careful to mitigate partial volume effects from orbital fat and air-filling in the paranasal sinuses Fig. b.25, including (1) point-specific parameters, which directly mirrored the T2 values: mean  and other cumulative T2 histograms: lower percentiles  , higher percentiles , where the nth percentile was the point at which n percent of voxel values were situated on the left area of the histogram27; and (2) histogram shape-related parameters, which indirectly reflected the heterogeneous distribution of T2: (a) standard deviation ; (b) skewness (reflecting asymmetry of the histogram distributions); (c) kurtosis (representing the peaked nature of the histogram); (d) entropy (describing irregularity or complexity of the distribution of a parameter of interest); and (e) inhomogeneity .Based on histogram analysis of the ROIs, T2 histograms and a variety of parameters were generated through combined analysis by Fire Voxel and SPSS software . These parameters were divided into two categoriesEOMs were divided into involved or normal-appearing in a binary manner. Involvement was visually evaluated as the presence of swelling and brightness signal intensity  on T2-IDEAL imaging. EOMs without these manifestations were designated as normal-appearing extraocular muscles (NAEOMs). The two neuroradiologists determined muscle involvement for TAO patients, but not for HCs. Disagreements regarding classification were resolved by a third neuroradiologist with 15 years of experience. The SIR and T2 parameters were classified based on the involvement evaluation.Data are expressed as the mean ± SD. A significance level of P < 0.05 was considered statistically significant. All statistical analyses were performed using the SPSS statistical software package.t-tests or paired Wilcoxon signed-rank tests were conducted to investigate changes in histogram parameters and SIRs of EOMs in patients who achieved good therapeutic responses.Interobserver agreement for histogram parameters and SIRs was assessed using the intraclass correlation coefficient. The Kolmogorov–Smirnov test was used to assess the normality of continuous variables with regard to the SIRs and histogram parameters. Based on the results of the Kolmogorov–Smirnov test, comparisons among the three groups were performed using the ANOVA or independent-samples Kruskal–Wallis test. Receiver operating characteristic (ROC) curves were constructed to evaluate the diagnostic performance of the SIRs and T2RT histogram parameters to differentiate the really normal EOMs and the early involved EOMs (that is NAEOMs). Paired This study was approved by the Institutional Review Board (IRB) of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, and we pledged to abide by the declaration of Helsinki (2000 EDITION) in accordance with the relevant medical research rules of China in the study. Written informed consent was obtained from all patients for MRI scanning, but the written informed consent of the data usage for this research was waived as the MRI protocol is conventional in our hospital and this is a retrospective study. All patient-sensitive information was kept with confidentiality and used only for the purpose of the study.All procedures performed in the studies involving human participants were in accordance with the ethical standards of our institutional review board and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.Written informed was accepted from all patient.


POLLED allele into a tropically adapted Australian beef cattle population via conventional breeding or gene editing (top 1% or 10% of seedstock bulls/year) for 3 polled mating schemes and compared results to baseline selection on genetic merit  alone, over the course of 20 years. The baseline scenario did not significantly decrease the 20-year HORNED allele frequency (80%), but resulted in one of the fastest rates of genetic gain ($8.00/year). Compared to the baseline, the conventional breeding scenarios where polled bulls were preferentially used for breeding, regardless of their genetic merit, significantly decreased the 20-year HORNED allele frequency (30%), but resulted in a significantly slower rate of genetic gain . The mating scheme that required the exclusive use of homozygous polled bulls, resulted in the lowest 20-year HORNED allele frequency (8%), but this conventional breeding scenario resulted in the slowest rate of genetic gain ($5.50/year). The addition of gene editing the top 1% or 10% of seedstock bull calves/year to each conventional breeding scenario resulted in significantly faster rates of genetic gain . Overall, our study demonstrates that, due to the limited number of polled Australian Brahman bulls, strong selection pressure on polled will be necessary to meaningfully increase the number of polled animals in this population. Moreover, these scenarios illustrate how gene editing could be a tool for accelerating the development of high-genetic-merit homozygous polled sires to mitigate the current trade-off of slower genetic gain associated with decreasing HORNED allele frequency in the Australian Brahman population.Dehorning is the process of physically removing horns to protect animals and humans from injury, but the process is costly, unpleasant, and faces increasing public scrutiny. Genetic selection for polled (hornless), which is genetically dominant to horned, is a long-term solution to eliminate the need for dehorning. However, due to the limited number of polled Australian Brahman bulls, the northern Australian beef cattle population remains predominantly horned. The potential to use gene editing to produce high-genetic-merit polled cattle was recently demonstrated. To further explore the concept, this study simulated introgression of the  Intact horns are extremely costly due to carcass bruising, hide damage, and danger to human handlers . Horn reThere are two different methods to remove horns depending on the age of the calf, disbudding and dehorning. In cattle, horns first form as a free-floating horn bud, usually evident at birth. If a calf is less than 8 weeks old, the unattached horn buds can be removed by disbudding via a hot iron or caustic paste. However, after this time frame the growing horn attaches to the skull and must be physically removed by dehorning using a scoop, gouge, saw, or wire. Amputating the fixed horn from the skull can expose the frontal sinuses, which is part of the cranial cavity that houses the brain, and typically results in larger and more invasive wounds . These wOver 170,000 calves are dehorned each year in Australia and almost 100,000 of those are in northern Australia  . In nortIn addition to the economic costs, common livestock management practices, such as dehorning, are under increasing public scrutiny . In respMoreover, dehorning in Australia is likely to be subject to stricter animal welfare legislation in the future . In 2014POLLED mutations have been identified in Bos taurus cattle on chromosome 1 (BTA1). The most common is a simple allele of Celtic origin (PC) corresponding to a duplication of a 212 bp sequence in place of a 10-bp deletion on chromosome 1 , and a 7-bp deletion and 6-bp insertion (P1ID) was identified through admixed Mongolian yaks (G), an approximately 110 kb duplication was revealed by genome-wide association studies of Nelore (Bos taurus indicus) beef cattle  cattle. Horns are inherited as an autosomal recessive trait . To datemosome 1 . Anotherome 1 F; . A thirdian yaks . A fourtf cattle .POLLED allele frequency of dairy cattle populations  has since increased considerably through systematic breeding, genetic testing, and the introduction of genomic breeding values in late August 2011. As a result, the proportion of polled cows in the Fleckvieh dual-purpose population is expected to be around 10.5% by 2021  such that they’re only about 5 years of genomic selection behind elite horned bulls . HoweverOn the other hand, there has been less of a need for investigating and modeling polled beef breeding schemes due to the availability of efficient beef producing genetics in homozygous polled beef breeds, like Angus. For example, in the United States there was a 58% reduction in beef calves born with horns from 1992 to 2007  and in EPOLLED mutation present in this breed is the PC allele, and that it has been introgressed from Bos taurus cattle  to produce two healthy, homozygous polled dairy bulls , while minimizing detrimental effects on inbreeding and genetic merit. In these simulation studies, gene editing reduced the undesired allele significantly faster than genomic selection or conventional breeding strategies alone. Additionally, both studies found that gene editing reduced long-term inbreeding levels in scenarios that placed all selection emphasis on the monogenic trait. Furthermore, these studies found that the addition of gene editing helped minimize the decrease in the rate of genetic gain resulting from selection emphasis on a monogenic trait.Our previous simulation study modeling the United States dairy cattle population  and a siPOLLED allele to the beef industry as compared to the dairy industry. For instance, the majority of dairy calves are produced via artificial insemination (AI)  , whereasion (AI) . Additioion (AI) . In contion (AI) .POLLED allele frequency in the northern Australian Brahman beef cattle population. In particular, we sought to compare current conventional breeding/selection strategies used in this population with the addition of gene editing. Additionally, we compared and contrasted the results of this beef population simulation to our previous United States dairy simulation results , over the course of 20 years.The objectives of this study were to (1) estimate the availability and genetic merit of polled Brahman bulls available in Australia, and (2) simulate introgression of the POLLED allele into the Australian Brahman population via conventional breeding or with the additional use of gene editing.Computer simulation using the program “geneedit”  was usedThe “geneedit” program simulates gene editing applied to a cattle population as an extension of Cole’s program (2015) to manage multiple recessives. Complete source code is available on GitHub and is in the public domain. The basic simulation procedures are identical to those described in detail by –5 is used, which results in “AA” genotypes being converted to “Aa” genotypes when calves are created. All base population animals are treated as founders and mated at random for 10 years to produce the population used for specific mating schemes.First, base bull and cow populations are formed by simulating animals with varying ages by drawing true breeding values (TBV) from normal distributions and randomly sampling horned genotypes from a Bernoulli distribution with a parameter equal to its allele frequency in the population of interest. The program ensures that at least one carrier bull and cow will be present in the base population for each recessive. Also, to prevent a minor allele from being lost due to drift a constant mutation rate of 10Each round of the simulation represents 1 year of calendar time, and generations overlap. The population size increases each round of the simulation until the user defined maximum population size of bulls and cows is reached. Each year, animals are culled for reaching the user defined maximum permitted age. Also, if the population is too large after age-based culling then cows will be culled at random (involuntary culls) and bulls with the lowest TBV or other user defined culling criteria will be culled.Any deviations from the basic simulation procedures described above, the specific mating schemes and gene editing strategy modeled in this Australian Brahman simulation study are described in detail in the section below and complete simulation parameters are listed in the The program is a stochastic simulation, but individual replicates can be recreated if the same seed is used for random variate generation. When the software was designed the goal was to examine changes in allele frequencies in cattle populations under different mating strategies, not to create a comprehensive general-purpose simulator for animal breeding. Because of that focus, selection is on TBV that are assumed known without error rather than estimated breeding values (EBV). This also means that genomic selection is not supported.A population of 10 nucleus (seedstock) herds supplying breeding bulls to 200 multiplier  herds was modeled. Ten replicates of each scenario were simulated for 20 years, with overlapping generations.All simulations were performed on a Thinkmate RAX QS6-4210  workstation with four 12-core AMD Opteron 6344 processors with a clock speed of 2.6 GHz, 512 GB of DDR3 1600 MHz RAM, and CentOS Linux EL7.POLLED allele frequency and the number of homozygous polled, heterozygous polled and horned sires were calculated from Australian Brahman animals with a SNP-based POLLED test (GeneSeek®) results from 2014 to 2017 , was calculated for each sire group as an indicator of overall genetic merit . to 2017 . It shou® SNP-based POLLED test data used to derive allele frequency and genetic merit estimates in this study may not be completely representative of the Australian Brahman population because the POLLED test is bundled with parentage and higher-density SNP genotyping for the Australian Brahman single-step genomic evaluation. There may have been a non-random selection of phenotypically polled animals submitted to determine if they were homozygous polled. However, this dataset included a large number of herds and different sires, so estimates based on this dataset were used for further modeling. All values are in Australian dollars. Two-tailed, unpaired student t-tests were used to determine if the average $JapOx value for each genotype was significantly different from one another. P-values of ≤0.05 were considered to be significantly different.The GeneSeekNorthern Australia is a vast region, covering approximately 400 million ha located within the subtropics and tropics. Major characteristics of this production system are low stocking rates , large management groups , multi-sire matings, and irregular handling and husbandry of cattle—typically only twice annually . In largB. indicus and B. indicus cross, primarily through the use of Brahman cattle  or −5 to 0 (bulls) by sampling from a uniform distribution. True breeding values ($JapOx) for each animal were determined by randomly sampling from a normal distribution, with a standard deviation (SD) of $34 for both the seedstock and commercial populations, and a mean of $34 for seedstock cows and $0 for commercial cows . Base poy of 80% . MoreoveThe flow of operations in the simulation is shown schematically in calf = 0.5(TBVsire + TBVdam) + MS, where TBVcalf, TBVsire, and TBVdam are the TBV of the calf, its sire, and its dam, respectively. The Mendelian sampling term was drawn from a normal distribution with a mean of 0 and a variance of fS and fD are the coefficients of inbreeding of the sire and dam, respectively, and Calves were born annually in the same herd as their dams. Sex was assigned randomly to new calves with a 50:50 sex ratio. The TBV for new calves were created by taking the parent average and adding a Mendelian sampling term (MS): TBVf $JapOx . Inbreedf $JapOx  using thf $JapOx . All basf $JapOx .In scenarios that included gene editing , it was New calves were subject randomly to a pre-weaning calf loss of 8% (seedstock) or 13% . Additionally, new horned calves of both populations were subject randomly to a dehorning mortality rate of 2% . RemovalSeedstock bull calves were kept for 1 year and then as yearling bulls they were sorted by $JapOx ranking (highest to lowest) and, depending on the mating scheme used, also by polled genotype. The seedstock population retained the top 5% of their yearling bulls (as defined by the mating scheme) for breeding to seedstock cows and the remainder of seedstock yearling bulls were moved to the commercial population for mating to commercial cows. In contrast, commercial population bull calves were all castrated and sold for beef annually, unless noted otherwise in a mating scheme.Animals were culled at the end of a simulated year. In both populations, cows were culled first by age (≥10 years) and then in order to not exceed a maximum populations size of 3,000 seedstock  and 100,000 commercial  females, they were also culled through random selection, which modeled involuntary culling. Bulls were culled first by age . Additionally, to maintain the allowable population size bulls were culled by the lowest $JapOx ranking and variably by polled genotype, depending on the mating scheme used, . The maximum allowable population sizes were 60 seedstock and 2,000 commercial mating-age-bulls .Four total mating schemes, one baseline (A), two polled preference (B and C) and one obligatory polled (D) were modeled . The thrFor retention in the seedstock population, baseline scheme A ranked the seedstock yearling bulls solely on $JapOx. Whereas polled mating schemes  ranked the seedstock yearling bulls first on polled genotype  and then on $JapOx. In baseline scheme (A) and polled preference schemes (B and C), all bulls used for breeding were sourced from the seedstock population.To establish a baseline and model current practice, scheme A used $JapOx as the sole sire selection criterion .In polled preference scheme B, polled bulls (both heterozygous and homozygous) were preferentially used for breeding, regardless of their genetic merit ($JapOx). If the maximum number of matings was reached for all polled bulls before all females were covered, horned bulls were used to cover the remaining females. Polled preference scheme C preferentially used only homozygous polled bulls for breeding and if needed, both heterozygous polled and horned seedstock bulls were used to cover the remaining females.In contrast, obligatory polled scheme (D) could only use homozygous polled bulls for breeding. If there were not enough seedstock homozygous polled bulls, then homozygous polled bulls from the commercial population were used to fill the deficit. As previously mentioned, the genetic merit ($JapOx) of these commercial bulls was on average one standard deviation less ($34) than seedstock bulls ($68). This scheme models what might be expected if producers are legally or contractually prohibited from using genetics that result in horned offspring.HORNED allele frequency, inbreeding, $JapOx, the total number of animals sold for beef per year, and the number and genotype of sires used per year were compared between all 10 scenarios. Additionally, Hardy–Weinberg principles were used to estimate the 20-year genotype frequencies based on the calculated HORNED allele frequencies of each scenario. Significance of the changes in HORNED allele frequency, inbreeding levels, the total number of animals sold for beef per year, and the number and genotype of sires used per year were determined using Tukey’s Honest Significant Difference test. To determine significance of changes in the rate of genetic gain, linear regressions of TBV for $JapOx on birth year were used. P-values of ≤0.05 were considered to be significantly different for all analyses. The results  result . The results consisted of 39 homozygous polled, 443 heterozygous polled, and 1,051 horned animals. The allele frequency was calculated to be 20% for the current Australian Brahman population. The 39 homozygous polled animals were sired by 25 different sires (not including unknown sires). Out of the tested animals, 1,174 were male and consisted of 32 homozygous polled, 360 heterozygous polled and 782 horned (P = 0.32 compared to horned) and $44.83 (P ≤ 0.05 compared to horned), respectively  of horned animals , 69.7% , and 71.4% , respectively  and 8.3% , respectively .The 20-year ectively . As a reectively . The addHORNED allele frequency of 6% . In scheme D only homozygous polled sires were used, thus conventional breeding and the addition of gene editing the top 1 and 10% of seedstock bull calves per year resulted in the same (P ≥ 0.9 compared to D) rapid decrease in HORNED allele frequency (The obligatory polled scheme D resulted in the lowest 20-year requency . Additiorequency .P ≥ 0.05 compared to A). The addition of gene editing the top 1% of seedstock bull calves per year to the polled preference mating schemes (B_1% and C_1%) increased 20-year inbreeding to 0.85% . However, the addition of gene editing in all other scenarios  resulted in similar 20-year inbreeding levels (P ≥ 0.05 compared to A). The inbreeding at year 20 based on an estimation of Nm is number of mothers, NF is number of fathers) was 0.22% for all scenarios, which is lower than that calculated by the simulation program due to the fact that the number of parents does not consider the relatedness between the seedstock animals as our simulation did. This in turn was higher than the inbreeding of 0.15% which would be expected under random mating.The seedstock population inbreeding levels inbreeding remained below 1% in all scenarios . In baseP ≤ 0.05 compared to A).Baseline scenario A, which placed no selection pressure on polled, resulted in a 20-year $JapOx value of $226 , respectively (P ≤ 0.05 compared to B) and $17 , respectively. Moreover, the addition of gene editing the top 10% of seedstock bull calves per year (B_10% and C-10%) further increased the 20-year $JapOx value by $31  and $29 , respectively. In fact, the 20-year $JapOx values in scenario B_10%  and C_10%  were not significantly different (P ≥ 0.9) from the baseline scenario A  and $198 . The 20-year $JapOx value in D_10% was similar  to the polled preference conventional breeding scenario B.The obligatory polled conventional breeding scenario D slowed the rate of genetic gain the most, resulting in a $178  20-year $JapOx value. The addition of gene editing the top 1 and 10% of seedstock bull calves per year to this scheme (D_1% and D_10%) significantly increased the 20-year $JapOx value by $10  and $15 , respectively (P ≤ 0.05) rates of genetic gain compared to the conventional breeding scenarios  of each polled mating scheme  seedstock homozygous polled sires used for breeding out of the 60 total sires . BaselinP ≤ 0.05) more homozygous polled sires used for breeding compared to A and 0 horned sires were used. Additionally, by year 10 all sires used for breeding were homozygous polled. However, the addition of gene editing the top 1 and 10% of seedstock bull calves per year to these polled preference schemes resulted in all sires used for breeding being homozygous polled by year 5. The greatest number of gene edited sires used for breeding was 15  around year 5 in scenarios B_1% and C_1%, whereas 55  were used around year 10 in scenarios B_10% and C_10%  resulted in 56  fewer gene edited sires being used for breeding in year 5 compared to B_1% and C_1%. Additionally, D_10% also resulted in 9  fewer gene edited sires being used for breeding in year 10 compared to B_10% and C_10%. Fewer gene edited sires were used in obligatory scheme D because there were more conventionally bred homozygous polled sires available due to the mating scheme design.Interestingly, the addition of gene editing the top 1% of seedstock bull calves per year to obligatory scheme D resulted in 5 , only 3  and 7  of the sires used for breeding were homozygous polled via gene editing, respectively  of horned animals , 49.1% , and 70.0% , respectively  and 28.9% , respectively  compared to conventional breeding scenarios, B and C, respectively. In contrast, the addition of gene editing the top 10% of seedstock bull calves per year (B_10% and C-10%) resulted in a 1.2%  and 1.5%  lower 20-year HORNED allele frequency, respectively  resulted in a similar 20-year ectively . These s year 20 .HORNED allele frequency of 8.0%  rapid change in HORNED allele frequency. Additionally, scheme D was estimated to result in the highest percentage of homozygous polled animals (84%) in year 20 (The obligatory polled scheme D resulted in the lowest 20-year  year 20 .P ≥ 0.05 compared to A) 20-year inbreeding levels of 0.005% . All of the obligatory polled scenarios, including the addition of gene editing 1 and 10% of the seedstock bull calves per year,  resulted in a significantly higher 20-year inbreeding level of approximately 0.012% . These values were slightly higher than the 0.006% 20-year inbreeding value estimated from only the number of parents in the population, which in turn was similar to the inbreeding value expected under random mating.The commercial population inbreeding remained below 0.02% in all scenarios . In baseIn year 1 of all scenarios, only commercial base population bulls were available for mating because the seedstock herds had not yet started supplying bulls to the commercial herds . BaselinAfter year 1, baseline scenario A and polled preference schemes B and C, only used bulls sourced from the seedstock population. In contrast, due to the limited number of homozygous polled sires, commercial bulls were needed to supplement the supply of seedstock bulls for all 20 years in obligatory polled scheme D. In this scheme, using a majority of commercial (∼900) and several seedstock (∼240) homozygous polled bulls, still resulted in a deficit of ∼350 bulls in year 5 .P ≤ 0.05 compared to A).After 10 years of baseline scenario A there was little change in genotype availability of sires, resulting in only 88  homozygous polled sires (4%) used for breeding compared to 1,217  horned (60%). In contrast, the polled conventional breeding scenarios  resulted in 612 , 685 , and 1,912  more homozygous polled sires used for breeding, respectively (P ≤ 0.05 compared to A) and 773  homozygous polled sires for breeding, respectively. The addition of gene editing the top 1% of seedstock bull calves per year (B_1% and C_1%) resulted in a similar number  of homozygous polled sires used for breeding in year 10. In contrast, the addition of gene editing the top 10% of seedstock bull calves per year (B_10% and C-10%) resulted in 107  and 89  more homozygous polled sires used for breeding in year 10, respectively  in year 10 and thereafter. However, ∼38%  of the bulls used for breeding in year 10 of the obligatory polled conventional breeding scenario D were commercial, which on average had lower genetic merit than seedstock bulls. The addition of gene editing the top 1% of seedstock bull calves per year to this scheme (D_1%) resulted in a similar number of commercial bulls used for breeding in year 10 . In contrast, the addition of gene editing the top 10% of seedstock bull calves per year to this scheme (D_10%) significantly decreased the number of commercial bulls used for breeding in year 10 by 71 .The obligatory polled scheme D clearly used the greatest number of homozygous polled sires  gene edited seedstock bulls, which was ∼4% of the total bulls used for breeding. By year 20 of these scenarios , only ∼1%  of the bulls used for breeding were gene edited seedstock bulls , used 73  compared to baseline scenario A , all mature females were mated, and ∼53,000 animals  were sold for beef per year . Strikinenario A .P ≤ 0.05 compared to A) animals sold for beef in year 10 (P ≤ 0.05) compared to baseline scenario A  numbers of total animals sold for beef per year for each respective polled mating scheme (P ≤ 0.05) animals sold for beef in year 20 than baseline scenario A  resulted in similar  held an open public consultation and found that issues related to “pain relief for surgical procedures”  were the most controversial for the Australian public . All aniPOLLED allele frequency is much higher in the Australian Brahman population  market pressure is placed on the Australian beef industry to eliminate dehorning. In our simulation results, both the seedstock and commercial population results for each scenario followed similar patterns. Although each scenario had a greater impact on the seedstock population due to its smaller size, an important measure of a seedstock population is its gene flow into the larger commercial population. Therefore, the following discussion will focus primarily on the commercial population results.HORNED allele frequency . This scenario resulted in little change in requency  or genotrequency . As mentrequency , strong HORNED allele frequency, although it may limit the number of cows actually available to breed in some scenarios and make little difference in others. For example, in scenario D, due to population size, there is no involuntary culling of cows until year 15, and by that time there are no horned animals remaining to cull.The baseline scenario A commercial population reached a 20-year $JapOx value of $160 $8/year; . The basBaseline scenario A demonstrates the trade-offs associated with the business-as-usual situation. While scenario A resulted in one of the fastest rates of genetic gain, this scenario also used the most horned bulls for breeding throughout all 20 simulated years  and consHORNED allele frequency to ∼30% by year 20.The polled preference scenarios were chosen to represent an intermediate situation where some producers prioritize using polled sires (scenario B) or choose to only use homozygous polled sires (scenario C) while other producers continue to select sires based solely on $JapOx. This situation may arise if AHA were to implement the public suggestion of “mandating pain relief irrespective of the age of the animal” for surgical procedures, such as dehorning . Both scHORNED allele frequency because there were not enough homozygous polled Holstein sires available  for scenario C to be successful. In contrast, there is a larger proportion of Australian Brahman homozygous polled sires available , so C was the optimal conventional breeding scenario for the Brahman population in terms of substantially decreasing HORNED allele frequency while sustaining the rate of genetic gain. This simulation finding is in agreement with Interestingly, the Australian Brahman homozygous polled-only preference scenario C results contrast with the same scenario modeled for the United States dairy population. In the United States dairy simulations, scenario C  did not significantly reduce the As demonstrated in polled preference scenarios, B and C, Australian Brahman producers currently have genetic options available to proactively address this animal welfare concern before it becomes a legal requirement. In our simulation there was a significant decrease ($1.30/year) in the rate of genetic gain in conventional breeding scenario B and C compared to the baseline scenario A. However, after 10 years of both B and C there was a benefit in that these scenarios had less dehorning-related calf loss resulting in over 200 more steers sold for beef annually as compared to scenario A.POLLED allele frequency through conventional breeding methods. Consistent with United States dairy findings  compared to the conventional breeding scenarios, scenarios B_1% and C_1% still resulted in significantly slower ($0.50/year) rates of genetic gain compared to baseline A. However, the addition of gene editing the top 10% of Australian Brahman seedstock bull calves per year did result in similar rates of genetic gain to baseline A. Therefore, the overall optimal Australian Brahman scenario based on maintaining the rate of gene gain and rapidly decreasing Obligatory polled scheme D modeled a case where market expectations or government policies force the Australian beef industry to completely ban dehorning, as previously advocated for by animal rights groups . This siWe chose to model the use of commercial homozygous polled sires to supplement the supply of seedstock bulls when necessary. This model could be achieved by separating and leaving intact  any commercial bulls without horns during the first calf gathering and processing. These phenotypically hornless commercial bulls could then be genomically tested to determine their polled genotype. The confirmed homozygous polled commercial bulls could be retained or sold for breeding while the heterozygous polled commercial bulls could be castrated and sold for beef as usual.HORNED allele frequency of less than 10%, but scenario D also had the slowest rate of genetic gain of $5.50/year, compared to baseline scenario A of $8.00/year. Although there was a significant difference between the Australian Brahman obligatory polled conventional breeding scenario D and baseline A rates of genetic gain (∼$2.50/year), the difference was much less drastic than between the similar United States dairy scenarios (∼$34.00/year)  . AdditioIn this simulation, once all the seedstock and commercial homozygous polled bulls reached their maximum 35 matings per year, the remaining females were left open. This model was chosen to demonstrate the current deficit of homozygous polled Australian Brahman bulls that would be needed to make this obligatory polled scheme D feasible. A more realistic alternative would be to use homozygous polled bulls from other less tropically adapted breeds. However, this alternative could result in increased animal welfare issues and higher levels of mortality due to ill-adapted sires and their resultant progeny .POLLED allele is introgressed via traditional crossbreeding  significantly increased the 20-year commercial $JapOx value by $8 ($0.40/year) and $15 ($0.75/year), respectively, compared to conventional breeding scenario D. Gene editing enables the production of high-genetic-merit homozygous polled sires in only one generation as opposed to the several generations of back-crossing necessary to recover the high-genetic-merit when the breeding . AdditioPOLLED allele frequency in the United States and European dairy populations, while minimizing negative impacts on inbreeding and genetic merit  to improve quantitative traits, by selecting and gene editing the best animals based on their breeding value . Even thin vitro embryo production and ET program  or that insert other genetic material into the cell, will continue to be regulated by the Office of the Gene Technology Regulator (OGTR) as Genetically Modified Organisms (GMO). This ruling may make it cost-prohibitive to use gene editing to introduce the POLLED allele into multiple Australian beef bulls and cattle breeding programs, due to the expense and uncertainty associated with the GMO regulatory approval process (C allele (C allelic introgression, which have a significantly reduced frequency of unintended genomic integration (In 2019 the Australian government announced they will not regulate the use of gene editing techniques in plants, animals and human cell lines that do not introduce new genetic material . However process . It shou process  in reseaC allele . To avoiegration . IrrespeCurrently, the global regulatory status of gene edited animals is uncertain, and is the primary concern for further investment and development of gene edited animals . EditingMany of these regulatory decisions have been made in the absence of engagement with the scientific community, industry, stakeholders or publics . In part® and the Australian Brahman Breeders Association Limited. Python programs and notebooks used for simulation and analysis of gene editing in cattle breeding programs can be found at https://github.com/wintermind/gene-editing.The collated Australian Brahman genotype and EBV dataset that underlies several of the simulation parameters used for this study is not readily available due to privacy restrictions. Requests to access the datasets should be directed to GeneSeekJC and MM authored and modified the “geneedit” software. MM performed all the simulations and data analysis with additional input from JC, DJ, and AVE. DJ facilitated obtaining access to northern Australian cattle data. NC and IR provided and collated genotype data and breeding values for Australian Brahman cattle. MM wrote the manuscript with input from JC, DJ, and AVE. All authors reviewed and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


N-acetylglucosaminidase activity, leading to increased levels of nondegraded heparan sulfate (HS). A mouse model has been useful to evaluate novel treatments for MPS IIIB, but has limitations. In this study, we evaluated the naturally occurring canine model of MPS IIIB for the onset and progression of biochemical and neuropathological changes during the preclinical stages (onset approximately 24–30 months of age) of canine MPS IIIB disease. Even by 1 month of age, MPS IIIB dogs had elevated HS levels in brain and cerebrospinal fluid. Analysis of histopathology of several disease-relevant regions of the forebrain demonstrated progressive lysosomal storage and microglial activation despite a lack of cerebrocortical atrophy in the oldest animals studied. More pronounced histopathology changes were detected in the cerebellum, where progressive lysosomal storage, astrocytosis and microglial activation were observed. Microglial activation was particularly prominent in cerebellar white matter and within the deep cerebellar nuclei, where neuron loss also occurred. The findings in this study will form the basis of future assessments of therapeutic efficacy in this large animal disease model.Mucopolysaccharidosis type IIIB  is an autosomal recessive lysosomal storage disorder caused by the deficiency of alpha- GAGs including HS are normally degraded by step-wise enzymatic modification removal of terminal sugars and sulfates at the non-reducing ends of GAG chains2.Therefore, individual deficiency in the enzymes involved in these catabolic pathways results in the lysosomal storage of non-degraded GAGs. Mucopolysaccharidosis type III is a heterogeneous condition comprised of four disorders in humans, MPS IIIA–IIID . Each MPS III subtype is characterized by mutations in one of four distinct enzymes, each with an obligate and non-redundant role in HS catabolism. Deficiencies in the activities of each of these enzymes result in the accumulation of a disease-specific HS moiety at the non-reducing end (HS-NRE)2. Unlike most MPS disorders, MPS III patients have severe central nervous system (CNS) symptoms with relatively few peripheral disease manifestations.Mucopolysaccharidosis type III (MPS III—Sanfilippo syndrome) is a group of autosomal recessive lysosomal storage disorders (LSDs), each caused by a specific enzymatic deficiency in the catabolic pathway of the glycosaminoglycan (GAG), heparan sulfate (HS)NAGLU gene, which encodes the enzyme alpha-N-acetylglucosaminidase 5. Patients appear normal at birth but present with early developmental delays followed by severe behavioural abnormalities and progressive neurodegeneration. Patients typically succumb to their disease in their mid-teens1.MPS IIIB accounts for approximately 30% of all MPS III cases and is caused by mutations in the 6, which display increased levels of HS and the MPS IIIB-specific HS-NRE in brain tissue8, and neuropathological findings similar to those described in the human disease, including increased microgliosis and astrocytosis in the brain9. Treatment of MPS IIIB mice with recombinant human NAGLU (rhNAGLU) or AAV9-murine Naglu resolved biochemical, pathological and behavioural manifestations of murine MPS IIIB11 .There is currently no approved treatment for MPS IIIB that targets the underlying disease. The challenge of developing effective therapies can be aided by the characterization of animal models that recapitulate important aspects of the human disease. A NAGLU deficient mouse (MPS IIIB mouse) model has been generated12. Specifically, large animal models of MPS IIIB have been described in emus13, cattle14, swine15, and canines, but only the canine model exists in a research setting suitable to evaluate a preclinical therapy16. A canine model of MPS IIIB has been described in the schipperke breed. Two dogs with naturally occurring MPS IIIB were found to have physical signs of and adult onset at approximately three years of age, of cerebellar disease including ataxia and tremor17. Gross pathological analysis of these dogs at 4.2 and 5.6 years of age revealed atrophy of the cerebellum with Purkinje cell loss, neuronal cytoplasmic vacuolation/granulation, and microglial vacuolation throughout the CNS, with somatic disease restricted chiefly to liver, kidney and tissue resident macrophages throughout the body17. The molecular defect in the canine NAGLU locus is involves the insertion of approximately 45 A residues flanked by an 11 base pair duplication of native sequence 5′ to the insertion16 in the last exon of the gene. Hence, while the initial clinical and molecular characterization has occurred, a longitudinal biochemical and histopathological assessment of the CNS disease in the current outbred colony of MPS IIIB dogs has not yet been conducted.Despite such advances in MPS IIIB mice, small animal models are limited by a relative lack of neurological complexity and differences in physiological dynamics, making assessments of potential treatments for human use difficult. Larger animal models can provide additional, complementary disease pathology information and are well-suited for confirming the safety and pharmacologic activity of potential therapeutics before initiation of human testing. These models better represent the human disease and response to therapy because of the greater genetic similarity to humans, outbred status, robust immune system, greater size (including brain size and complexity), and longer lifespan. Large animal models of the MPS disorders have long been a productive focus of preclinical research in these disordersIn this study, we describe the onset and progression of biochemical and neuropathological changes in the CNS of MPS IIIB dogs and unaffected control dogs at different ages during development up to 20 months of age, before the time point when these dogs develop clear motor signs. The biochemical characterization of the kinetics of substrate accumulation focused on the cortical grey matter and the cerebrospinal fluid (CSF). Histopathological analyses centred on the cerebellum as a known area of disease pathology. Other brain regions associated with the symptomatology of human MPS IIIB were also examined including the hippocampus, thalamus, and hypothalamus. In addition to obtaining baseline natural history data to document progressive biochemical and neuropathological changes in preclinical MPS IIIB dogs, our goal was to determine measures that could inform therapeutic benefit of potential future treatments for MPS IIIB disease.The accumulation over time of non-degraded HS polysaccharide and oligosaccharide chains was quantified in MPS IIIB dogs at 1, 2, 3, 6, 9, and 12 months of age. Total HS, as defined by the sum of the internal disaccharides of HS, and the MPS IIIB-specific non-reducing end of HS (the substrate of NAGLU enzymatic activity) were measured in brain tissue , CSF, plasma, and urine  as well as the abundance of N-acetylated glucosamine. We found that the abundance of 2-O sulfated and N-sulfated HS subtly increased while the abundance of N-acetylated HS subtly decreased over time in brain tissue of MPS IIIB dogs  has been used as a surrogate marker of lysosomal storage burden and has been shown to indicate the size of the lysosomal compartment, despite only being a proxy measureols Fig. . Due to ols Fig. . A denseIn the cortex of 15-month-old MPS IIIB dogs, immunostaining for LAMP1 varied in intensity across the cortical laminae, and at different levels of the brain Fig. . RostralHippocampal neurons displayed subfield-specific LAMP1 staining in both unaffected and MPS IIIB dogs. This immunoreactivity was much darker in pyramidal neurons of CA3 rather than CA1 of MPS IIIB dogs and also within granule neurons of the dentate gyrus Fig. . In the 9. Given that mice may not live long enough to develop the full extent of cortical pathology, we evaluated the presence of cortical atrophy in MPS IIIB dogs by measuring cortical thickness in the most severely affected MPS IIIB dogs ≥ 20 months of age. Measurements were obtained from the somatosensory cortex, a representative region of the sensory cortex that is most severely affected in large animal models of similar neuropathic lysosomal disorders19. There was no significant difference in the thickness of the rostral suprasylvian gyrus  and astrogliosis (cerebellum). The distribution of these changes is similar to that of histological changes seen in the brain of an MPS IIB patient17.Quantitative analysis revealed clear signs of increased storage burden in cerebellar cortical layers of MPS IIIB dogs in addition to DCN and cerebellar white matter. Taken together these observations indicate that the DCN are the primary pathological target within the MPS IIIB cerebellum, exhibiting pronounced neurodegeneration and atrophy of surrounding white matter tissue.Pathological evaluation of the cerebellum confirmed it to be the most severely affected brain region in preclinical MPS IIIB dogs, displaying neuropathological changes that are evident relatively early in disease progression. One of the most prominent and previously unreported findings within the cerebellum was a substantial 50% loss of DCN neurons. This loss occurred relatively early in disease progression (present by 9 months of age) and did not appear to progress further, indicating that the majority of DCN neurodegeneration occurs in early stages of the disease. As such, it may be important to look in patients for symptoms that could potentially be related to this DCN loss. There was also significant cerebellar white matter atrophy in preclinical MPS IIIB dogs, which had almost half the volume compared to unaffected dogs. This may be due to axon loss or a demyelination process resulting from a degeneration or dysfunction of oligodendrocytes or defective myelination during post-natal development. However, we observed an increase in white matter volume in unaffected dogs suggestive of ongoing myelination during this time in development. The degree of Purkinje cell loss in these MPS IIIB dog samples was relatively small and not significantly different from controls, as was previously reported in older animals17, our current findings provide the first evidence for pronounced pathology within deeper structures of the cerebellum that include the white matter and DCN.The time-dependent accumulation of HS in the forebrain and progressive increase in LAMP1 staining in both the forebrain and cerebellum in MPS IIIB dogs indicate that MPS IIIB disease pathology clearly occurs prior to the onset of clinical signs in animals. Such signs only occur subsequent to 20 months of age, with all but the most severely affected dogs examined in this study being younger than this. While disease-related pathology is present throughout the brain, the cerebellum is more dramatically affected than other regions in preclinical MPS IIIB dogs. Although the loss of Purkinje neurons has been documented previously in the schipperke MPS IIIB dogs27. Similar symptoms include motor dysfunction, aggressiveness, hyperactivity, and language and communication deficits27. In addition to changes in brainstem volume and cortical regions, autism is associated with cerebellar atrophy and dysfunction, which may cause motor dysfunction as well as some non-motor symptoms such as deficits in language, communication, social interactions, stereotyped behavior, and cognitive dysfunction29. Similar symptoms are observed in MPS IIIB patients, suggesting that the cerebellar pathology seen in the MPS IIIB dogs may have relevance for early stages of human MPS IIIB disease. Later stages of the human MPS IIIB disease, which ultimately result in a vegetative state, are likely to involve more widespread neurodegeneration.While human MPS IIIB disease is thought to primarily affect cortical brain regions, the extent to which the cerebellum is affected and plays a role in the disease symptoms has not been thoroughly investigated. Interestingly, early stage MPS IIIB disease shares many symptoms with autism despite the different etiologies and MPS IIIB is often misdiagnosed as autismOur study reveals the dynamics of the onset and progression of neuropathology in this large animal model of MPS IIIB disease, before the onset of disease signs. We describe a series of markers that can be used for assessing therapeutic benefit in future studies, focusing on well-defined neuropathological and biochemical endpoints including the direct measure of NAGLU substrate accumulation (HS and HS-NRE), Iba1 as a marker of microglial activation, and LAMP1 as a marker of lysosomal enlargement.17. Animals were genotyped by 7 days old with polymerase chain reaction at the canine NAGLU locus, and enzyme deficiency was confirmed by testing their serum enzyme levels30. The MPS IIIB dogs and their unaffected counterparts were produced and maintained at Iowa State University’s Department of Animal Science according to guidelines of the USDA and NIH, and under approved protocols of the ISU IACUC. Animals were kept with their dams until weaning, and at 4 months segregated by sex. After weaning, dogs were fed ad libitum. CSF was aseptically collected under full anaesthesia by puncture of the cisterna magna after which dogs were euthanized by intravenous administration of sodium pentobarbital at approximately 1, 2, 3, 6, 9, 12, 15, and 20 months of age, after which encephalectomy was performed by Iowa State University Department of Animal Science staff. The numbers of individual dogs analysed at each age is summarized in Table The outbred MPS IIIB canine colony was founded based on a naturally-occurring mutation in schipperke dogs31. Briefly, HS from the samples was purified by anion exchange chromatography and digested with heparan lyases (IBEX Technologies). The HS fragments were tagged with isotropically labelled aniline by reductive amination and quantified by LCMS. Total HS  and the MPS IIIB-specific HS-NRE  are expressed as pmol/mg brain wet weight, pmol/0.6 μl CSF or plasma, or pmol/nmol creatinine in urine.Total HS and MPS IIIB-specific HS-NRE in homogenized brain tissue , CSF, serum and urine from approximately 1, 2, 3, 6, 9, and 12-month-old unaffected or MPS IIIB dogs  were quantified using the HS-NRE liquid chromatography-mass spectrometry (LCMS) assay (SensiPro) as described previouslyBrains were stored in formalin for extended periods before histology was performed. Before beginning histology, brain hemispheres were submerged in cryoprotectant, 30% sucrose in Tris-buffered saline  for at least two weeks. The forebrain was then separated from the cerebellum and cut perpendicular to the long axis of the brain into three rostrocaudal blocks that were sectioned individually. Blocks were coated with Lipshaw M-1 cryomatrix resin  and the forebrain cut coronally on a freezing microtome at 50 µm , with the cerebellum cut sagittally. Sections were collected into 24 well plates containing a lab-derived cryoprotectant solution , and stored at 4 °C until staining was performed.32. Briefly, every forty-eighth 50 µm section through each block was mounted onto microscope slides previously double coated with gelatin-chrome allum (ThermoFisher Scientific). After air drying for at least 24 h, sections were incubated in 0.025% cresyl violet  and 0.05% acetic acid  at 56 °C for 30 min. Sections were then differentiated through a series of graded concentrations of industrial methylated spirits  and cleared in xylene (VWR) for at least 24 h before coverslipping with DPX (VWR), a xylene-based mountant.In order to visualize tissue cytoarchitecture, regularly spaced sections were Nissl stained with cresyl violet, as described previously33, for the markers LAMP1  and Iba1 , in addition to GFAP  in the cerebellum. Briefly, after quenching endogenous peroxidase activity in 1% H2O2 (Fisher Scientific) for 25 min, sections were rinsed, and to reduce non-specific protein binding were incubated with 15% normal serum (serum from the host species of the secondary antibody) in TBS-T (TBS containing 0.3% IBA/v Triton X-100) for 30 min. Primary antibody was applied in a 10% serum solution in TBS-T , and left to incubate overnight at room temperature. After rinsing, sections were incubated with secondary antiserum  in TBS-T with 10% normal swine or rabbit serum, respectively, for 2 h. After rinsing with TBS, sections were incubated overnight with avidin–biotin-peroxidase complex . To visualize immunoreactivity, slides were incubated in a 0.05% solution of 3, 3′- diaminobenzidine tetrahydrochloride  with 0.001% H2O2 solution in TBS for approximately 8 min. The reaction was stopped with ice-cold TBS. After rinsing with TBS, sections were mounted on Superfrost Plus microscope slides (ThermoFisher Scientific), and air dried overnight. Slides were then cleared with xylene, and coverslipped with DPX (VWR).To assess lysosomal storage and microglial activation in the forebrain and cerebellum, regularly spaced sections were selected, and immunohistochemically stained, using standard protocolsStereoInvestigator software . These measurements were made in the primary somatosensory cortex, the rostral suprasylvian gyrus; this region was identified according to neuroanatomical landmarks34. Three sections containing this region were selected from each brain, and slides were blinded to age and treatment before analysis started. Ten measurements of cortical thickness were made per section, and these measurements were made in areas as far from gyrus curvature as possible to minimize variability. The mean regional cortical thickness was then calculated for each brain.To assess potential differences in cortical thickness, Nissl-stained sections were measured perpendicularly from the pial surface to white matter border, and this thickness was analyzed with 35 and was less than 0.08 in all these analyses. The number of Purkinje neurons was counted manually and expressed as the number per unit length within the same folium in each brain.Optical fractionator counts of the number of Nissl-stained DCN neurons were made using an appropriate grid size and dissector frame. Only neurons with a clearly identified morphology that fell within the dissector frame were counted. For all optical fractionator estimates, the mean coefficient of error of individual estimates was calculated according to the method of Gundersen and JensenStereoInvestigator software, and the mean microglial area determined ± standard area of the mean (SEM).The mean size of Iba1-stained microglia in the forebrain was obtained by measuring the cross-sectional area of at least 100 morphologically identified Iba1-stained microglia in each animal using Image-Pro Plus 5.0 software  was used to analyze the images with an appropriate threshold selected to distinguish the foreground immunostaining above the background. Macros were recorded to transfer the data to a spreadsheet for statistical analysis. The data obtained from the thresholding analysis was plotted graphically as a mean percentage area of immunoreactivity per treatment group ± SEM.The expression of GFAP, Iba1, and LAMP1 was measured in the cerebellum by semi-quantitative image thresholding analysis of each antigen. From three consecutive immunostained sections, 10 non-overlapping images were captured from each section for each of the areas examined. All images were captured at 40X objective by a live video camera  mounted on Zeiss Axioplan universal microscope (Germany). The lamp intensity, video camera setup, and calibration were kept constant throughout the image capturing process. p value less than or equal to 0.05.All statistical analyses were performed blinded to genotype and age, which was only revealed once all morphological analyses were completed. A Kolmogorov–Smirnov test, a nonparametric test which assesses the empirical distribution functions of two samples, was applied to measured microglial cell body areas . A t-test was used to determine whether there was a statistically significant difference between genotype groups in cortical thickness of somatosensory cortex. Results were considered statistically significant if they produced a Supplementary Information.


Offspring of these mothers as well as from control mothers were also examined. We were primarily interested in detecting whether cognitive deficits were present in the mothers and offspring in the long term and used a spatial learning and memory task as well as an object recognition task at three timepoints: 3, 8, and 12 months post-partum or post-natal, while measuring blood pressure and performing urine analysis after each timepoint. While we did not find significant deficits in preeclamptic mothers at the later timepoints, we did observe negative consequences in the pups of R+A+ mice that coincided with hemodynamic alterations whereby pups had higher whisker-evoked oxygenated hemoglobin levels and increased cerebral blood flow responses compared to control pups. Our study provides validation of this preeclampsia mouse model for future studies to decipher the underlying mechanisms of long-term cognitive deficits found in offspring.Preeclampsia is a common hypertensive disorder in pregnant women and whose causes and consequences have focused primarily on cardiovascular outcomes on the mother and offspring, often without taking into consideration the possible effects on the brain. One possible cause of preeclampsia has been attributed to alterations in the renin-angiotensin system, which has also been linked to cognitive decline. In this pilot study, we use a transgenic mouse model that chronically overexpresses human angiotensinogen and renin (R Preeclampsia is a common hypertensive disorder in pregnant women, with a worldwide incidence rate of just under 3% but varying between 2.7 and 8.2% depending on the region studied  and this+A+ females) that are chronically hypertensive. This is a realistic disease model of preeclampsia superimposed on chronic hypertension, not only because chronic hypertension represents a major risk factor for developing preeclampsia during pregnancy, but because these mice mimic several aspects of preeclampsia, including aggravated hypertension during pregnancy, glomerular endotheliosis, proteinuria, placental necrosis, lower pup weights, impaired angiogenesis of uteroplacental vessels, and increased circulating anti-angiogenic factors  of control females  and transgenic females with the R+A+ genotype, predisposed to become preeclamptic during pregnancy, entered the study. Baseline blood pressure (BP) was measured, and urine samples collected to assess proteinuria prior to mating. All females were then paired with a wild type (C57BL/6 background) male for breeding, and a female was deemed pregnant by the presence of a vaginal plug and later confirmed by weight gain. After 2 months of mating 13/15 control females became pregnant (average litter size of 5.69 ± 0.47), while only 8/15 R+A+ were pregnant (average litter size of 6.38 ± 0.65). Fifteen and eighteen days following the initial plug, a urine sample was collected, and BP was measured; this was also performed 5 days after giving birth, at which point pups were weighed. Urine samples and BP were taken in mothers 3, 6, and 12 months postpartum (PP). Cognitive testing on mothers was performed at 9, 14, and 18 months of age  alongside a subset of their offspring . Likely due to R+A+ females being chronically hypertensive, we found a higher death rate over time in this group, leaving us with n = 5 at study endpoint (12 months PP). Due to differences in cognitive function in offspring at 12 months of age and sufficient group sizes, we used optical imaging of intrinsic signals (n = 6/group) to asses cerebral hemodynamic function. Experiments were approved by the Animal Ethics Committee of the Montreal Neurological Institute  and complied with the Canadian Council on Animal Care.Mice were produced by breeding transgenic male mice heterozygous for human renin (Ren9 line) with female mice heterozygous for overexpressing human angiotensinogen (204/1 line) . An equaUrine samples (∼100 μL) were collected in 1.5 mL centrifuge tubes from mothers prior to being impregnated (baseline), and then after 15 days of gestation (day 1 defined by the presence of a plug), 18 days of gestation, 5 days PP and before the first behavioral test, 3 and 8 months PP. Samples were kept at −80°C, and then thawed and diluted 1:10 before measurements were made in duplicate. Proteinuria was determined as the ratio of albumin:creatinine using Albuwell and Creatinine companion mouse ELISA kits  according to manufacturer’s protocol.n = 10 mice/group) after each behavioral timepoint in mothers, and at 6 months of age in a subset of offspring using non-invasive tail-cuff plethysmography (Kent Scientific Company). Mice were habituated to the restraining device and tail cuffs for 10 min/d for 3 days before measurements were recorded. Ten additional acclimation cycles were performed before acquiring five measurements of systolic, diastolic, and mean blood pressure. This was performed 1 week prior to each behavioral testing session.Blood pressure was measured  to the NOR testing arena (45 cm wide × 45 cm long × 50 cm high). The following day, mice underwent both the familiarization and test phases. During familiarization, each mouse was given 5 min to explore 2 identical objects placed in opposite corners of the arena, equidistant from the center. Two hours after familiarization, one of the objects was replaced with a novel object and another 5 min exploration period was allotted. Both the location of the novel object and the objects were counterbalanced. Behavior was recorded using iSpy software and ODLog to record the amount of time spent exploring each object. An investigation ratio was calculated by dividing the time spent with the novel object by the total time investigating both objects during the test phase. This test was conducted at 3 separate time points using different pairs of objects at each time.n = 6/group, 12 months) were implanted with a cranial window made of a cover glass glued on a thinned bone preparation over the left barrel cortex and allowed 2 weeks to recover before imaging. Mice were anesthetized  and fixed with ear bars on a physiological platform  with online recording of body temperature, heart rate and respiratory rate. The platform was then moved to the imaging system ; the mouse cranial window placed parallel to the camera. Whisker-evoked changes in total, oxy- and deoxyhemoglobin  were measured with three light emitting diodes  placed 8 cm from the cranial window. Whisker-evoked changes in cerebral blood flow (CBF) were measured with laser speckle contrast imaging using a 780 nm laser diode. Images were acquired at a frame rate of 30 Hz in total (7.5 Hz for each wavelength), with a 14 ms exposure time. Each recording consisted of 7 blocks of right whisker stimulation . We included a jitter of 3 s to avoid habituation to the stimulation timing for the mice. Data were lowpass filtered at 0.3 Hz to suppress high frequency noise (mouse respiration) and analyzed using MATLAB to obtain whisker-evoked changes in cerebral blood volume and CBF from baseline. Analysis was as described in 0), and a pseudo-inverse and the modified Beer–Lambert law were used to extract relative changes in HbR and HbO. CBF was computed by quantifying the spatial contrast during laser illumination, defined as the ratio of the standard deviation to the mean intensity in a given spatial area. For each animal, the spatial location of the response in the contralateral barrel cortex was first identified. A region of interest around the maximum response was manually delineated avoiding medium, large arteries, and veins, and the signal averaged over all pixels to recover an impulse response function for each component: HbO, HbR, and CBF.A subset of pups . These hypertensive pups were examined with caution following all measures, but they did not significantly differ from normotensive pups birthed by the same R+A+ mothers. A cardinal feature of preeclampsia is the presence of proteinuria during pregnancy, assessed here by the ratio of albumin to creatine  but also PP, spiking sharply at 5 days PP (p < 0.001) and remaining elevated at 3 months PP (p < 0.01), and even more so at 8 months PP (p < 0.001), prolonged or persistent proteinuria has been found as long as 2 years after delivery in preeclamptic women (p < 0.01) than those from control mothers  and continued elevated BP levels when measured PP . The majcreatine , which nic women . Another mothers .p < 0.05). Despite a trend (p = 0.09) this deficit was no longer present at 8 months PP (p < 0.05) at 8 months, and not only did this deficit persist but also worsened when tested at 12 months of age .When tested on the MWM for spatial learning and memory, a primarily hippocampal-dependent task, preeclamptic mothers did not differ from control mothers on the visible platform training days (days 1–3) 3 months PP, indicating that they were capable of performing the task. However, when the platform was hidden, preeclamptic mothers took significantly more time to locate the platform on days 4–6  before lonths PP  nor at 1onths PP . Howevert(10) = 4.51, p < 0.01] in the offspring of preeclamptic mothers, largely driven by an increase in oxygenated Hb (HbO) by nearly twofold: HbO during whisker-stimulation of pups from preeclamptic mothers being 4.16 mM ± 0.17, and 2.50 mM ± 0.30 from control pups t(10) = 4.22, p < 0.01. No differences in deoxygenated Hb (HbR) were observed between groups. These findings are consistent with whisker-evoked increases in CBF driving the surge in HbO, corresponding to fresh supply of arterial blood, with HbR, typically in veins, being washed out of the activated region, as illustrated by the decreased HbR  in pups from preeclamptic mothers compared to that elicited in the offspring of control mothers  were markedly low during a preeclamptic pregnancy. One group has used PGF knockout mice as a preclinical model for offspring of preeclamptic mothers and found working memory deficits along with increased vascular density and length at the capillary level, without any changes in cerebral blood volume . Further+A+ preeclamptic mothers. Interestingly, while we found an enhanced hyperemic response, hyperperfusion has been observed in preeclamptic patients and has been linked to decreased vascular resistance, which can further lead to disruptions of the blood brain barrier and negative neurological outcomes (The regulation of CBF is altered by pregnancy, and even further by hypertension; both hypo- and hyperperfusion have been observed in preeclamptic mothers , and thioutcomes . While toutcomes . It is toutcomes , then gloutcomes . Due to There exists a tight communication between neural cells and blood vessels termed neurovascular coupling, and here we observe an altered hemodynamic response whereby too much blood is being sent to the activated somatosensory cortex. We found that oxygenated hemoglobin levels were elevated in response to the same sensory stimulus in offspring of preeclamptic mothers. Capillaries are comprised mainly of endothelial cells, which play an important role in neurovascular coupling , and areIn this pilot study we used the pups from an established model of preeclampsia and validated their use for future studies as a realistic model of offspring of preeclamptic mothers in terms of their cognitive outcome. Our findings are in line with cognition being altered in preeclamptic mothers in the short-term without any lasting deficits . They alThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.The animal study was reviewed and approved by the Animal Ethics Committee of the Montreal Neurological Institute.LT assisted in the design of the study, conducted all behavioral experiments, measured blood pressure and took urine samples at all time points excluding baseline, analyzed data, and wrote and finalized the manuscript. CL implanted mice with cranial windows and performed optical imaging experiments and analysis. JR assisted in the study design and acquired baseline urine and blood pressure measurements. JL provided the mouse model used in this pilot study and provided equipment for urine analysis to assess proteinuria. FL provided their expertise and codes for analysis of optical. LP helping to initiated the study and contributed to the editing of the final manuscript. EH supervised the project, assisted in the design of the study, contributed to the writing, and editing of the final manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


According to the voxel measurements after treatment with MagdentMed™ pulse electromagnetic healing caps, there was a significant difference p < 0.0001 between bone radiodensity before treatment 310.7 ± 53.26 SD and bone radiodensity after treatment with MED caps 734 ± 61.96 SD. The most common result of our study was a slightly higher radiodensity nearest the interface of dental implants after treatment.As a result of the loss of a tooth, there is a decrease in trabecular bone and loss of height and width of the adjacent bone. This study was designed as an observational imaging study, regarding structural changes that may occur during healing after the placement of Titanium dental implants. For this purpose, Cone Beam Computed Tomography was used in order to determine bone modifications around dental implants, loaded either with conventional healing caps or with healing caps pulsating electromagnetic waves, Magdent™, Haifa, Israel. The mean age of the study population was 49.84 ± 3.29 years : 46.55−53.13). According to the voxel measurements after conventional treatment, there was a significant difference  The human periodontium is a dynamic complex of tissues that undergoes remodeling in the area after the extraction of a tooth, because the alveolar bone requires mechanical stimulation to maintain its shape and density. Following the loss of a tooth, there is a decrease in trabecular bone and loss of height and width of the surrounding bone. Bone loss has been well documented by many researchers in human models ,2,3,4 anThe quality of bone depends on the anatomical location, age of the patient, general health status, and local infections and tumors that may influence the quality of the bone. The most dense bone type is found in the anterior part of the mandible, followed by the posterior part of the mandible, the anterior part of the maxilla, and the posterior part of the maxilla. Assessment of bone density was difficult in the past, but an important advancement in bone quality estimation was made using computed tomography (CT), by characterizing bone density in terms of the CT number (or radiodensity), expressed in Hounsfield units (HU) . The CT This study was designed as an observational imaging study, regarding structural changes that may occur during healing after the placement of dental Titanium implants. For this, Cone Beam Computed Tomography was used in order to determine bone modifications around dental implants, loaded either with conventional healing caps or with healing caps pulsating electromagnetic waves, . The study was planned as a split-mouth controlled study, on 29 patients in need of oral rehabilitation based on dental implants, using the following protocol:(1)Age between 30–60 years;(2)Kennedy class I, II or III edentation;(3)Healthy subjects.The inclusion criteria for the treatment were:(1)Poor oral hygiene;(2)Smoking;(3)The presence of systemic diseases that could affect the outcome of surgical treatment;(4)Subjects suffering from diabetes.The exclusion criteria were:All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation  and with the Helsinki Declaration of 1975, as revised in 2008. Informed consent was obtained from all patients for inclusion in the study. The Ethics committee of Algocalm Private Medical Center of Târgu Mureș, Romania, approved the study (898/29.05.2020).In the subsequent stage of the study, the clinical objectives were met, namely the determination of the radiological bone level, width, height and bone density on Cone Beam Computed Tomographyscans . CBCT images were recorded using a tube voltage of 89 kV and a current intensity of 6 mA, the images were recorded with a cylindrical field of view (FOV) of 82 mm both in diameter and height , the voxFor image acquisition, we used a ProMax 3D CBCT unit , with the previously mentioned settings. Images were acquired and saved in JPEG format. To locate implant target sites, bone density, and to record the mean CT number of the trabecular bone from these sites, we used the OnDemand 3Ddata App™ software . In each site, our region of interest was a square volume of bone located within the alveolar ridge . All datEach voxel of the CBCT volume is characterized by a CT number, expressed in HU (Hounsfield Units). The software displays the mean value of the CT numbers of the constituent voxels and the standard deviation of these CT numbers .After recording the preliminary clinical situation, 8 patients from the study group (27.5%) underwent fixed orthodontic treatment for space opening in order to insert implants in the best possible position. Fixed orthodontic treatment varied from 8 weeks to 17 weeks and the medium space gained was approximatively 2.3 mm. In the subsequent stage of the study, the patients underwent initial periodontal treatment, including manual and ultrasonic debridement and cleaning, training on the correct brushing techniques, and the use of oral hygiene aids.(1)Infiltration.Peripheral trunk anesthesia.Anesthesia of the concerned area by:(2)Incision: a horizontal incision on the alveolar ridge package ± 1–2 vertical incisions, starting from the horizontal one towards the bottom of the vestibular mucosa.(3)Detachment and reflection of the mucoperiosteal flap on the vestibular and/or oral face of the tooth; to maintain the viability of the flap, saline irrigations were performed.(4)Insertion of MIS Seven™ dental implants, MIS Dental Implants, Israel, standard platform, according to the bone characteristics for each individual site, based on the CBCT scans. After insertion, a resonance frequency analysis RAF was performed using an implant stability tester Osstell™ ISQ . Implants stability ranged from 58 ISQ to 74 ISQ values on the tester.(5)Loading of dental implants either with conventional healing caps, or with pulsating electromagnetic caps  .(6)Repositioning and suturing the flap.After re-evaluating the subjects 6 weeks post-sanitization and confirming the need for surgical therapy, all patients underwent the following surgical protocol:(1)Patients were instructed to use an antiseptic mouthwash daily for 3 weeks  and antibiotic therapy was instituted;(2)The sutures were removed when the postoperative wound was clinically healed and when they were no longer needed to keep the flap in place (7–10 days);(3)Patients were advised not to brush the area until 2 weeks postoperatively, professional brushing was performed if necessary; they were also recommended a diet that did not require dental units for hard chewing;(4)At 2 weeks, the patients were retrained regarding dental brushing techniques, including interproximal brushing;Postoperative indicationsIn the next phase of the study, immediately after implantation, healing caps were placed in position in order to achieve the best healing possible. The study group was divided into two sub-groups as follows:Subgroup 1 consisted of 28 dental implants placed in all 12 patients included in the study, covered with conventional/standard healing caps , and subMagdent’s™ MED (MagdentMed) novel technology utilizes electromagnetic fields to stimulate, accelerate, and improve bone formation and quality for more successful dental implant procedures in shorter time. MED consists of a battery, an electronic device, and a coil that fits most implant models in much the same way as current simple/conventional healing abutments.After 60 days of healing process, new CBCT images were recorded in order to assess bone density changes that might have occurred near the implant sites .In the next phase of the study, after a healing period of 60 days, new measurements regarding bone density were recorded . To avoit test for unpaired (independent) and paired (dependent) data. The Kolmogorov–Smirnov test was used to test the normality of the data. The chosen significance threshold was alpha = 0.05, considering p significant when p < 0.05.Statistical processing was performed using GraphPad Prism™ V6.01 software for Windows™ . Statistical analysis involved the use of the Student’s Twenty-nine patients  were enrolled in the study and all of them showed modifications regarding bone density around dental implants. The mean age of the study population was 49.84 ± 3.29 years : 46.55–53.13). p < 0.0001 between bone radiodensity before treatment 288.1 ± 47.16 Standard Deviation (SD) and bone radiodensity 688.1 ± 81.02 SD after treatment.According to the voxel measurements after treatment with conventional caps , there wp = 0.59 between the 2 study groups; 697.2 ± 91.44 SD versus 679.7 ± 75.40 SD treated with conventional healing caps, regarding age  745.9 ± 64.05 SD versus study group 2 (45–60 years old) 684.5 ± 74.23 SD. Sub-analysis of the radiodensity in patients treated with pulse electromagnetic healing caps regarding age , showed p = 0.10 were recorded between voxel value regarding bone radiodensity before treatment either with conventional caps 288.1 ± 47.16 SD or pulse electromagnetic healing caps; 310.7 ± 53.26  and bone radiodensity 471.3 ± 60.99 SD after treatment with conventional healing caps of patients with orthodontic therapy in medical history. According to the voxel measurements after treatment with conventional healing caps , there wp < 0.0075 between bone radiodensity before implant placement 319.2 ± 51.44 Standard Deviation (SD) and bone radiodensity 514.4 ± 100. SD after implant placement with MED healing caps of patients with orthodontic therapy in medical history. According to the voxel measurements after treatment with MED healing caps  there waThe use of the HU to measure tissue density has aided radiologists in the interpretation of images and diagnosis of disease. One of the earliest uses of the HU as a quantitative measurement came in the evaluation of solitary pulmonary nodules. More recently, HU measurements of bone on quantitative CTs and conventional CTs have helped to determine bone mineral density. Hounsfield unit measurements of bone have also been the object of recent study to estimate bone quality .Lang et al.  reportedIn agreement with established evidence, the authors noted that the mandible generally had a higher cancellous radiodensity compared with the maxilla, and that certain combinations of bone quantity and quality were often seen together. Sixty of these observations have since been corroborated independently . It appeFurther studies  regardinOnly long-term randomized controlled clinical trials can give a definitive answer on the best way to treat lost implants. The long-term success of an implant depends on the regular maintenance schedule. During the maintenance phase, the perimeter tissue should be evaluated for inflammation. X-rays will show the bone issues around the implants. To date, no methodology has been established as a gold standard for the most correct use of gingival healing caps .Controlled trials combined with comparative randomized clinical trials of healing caps pulsating electromagnetic waves are limited in number and have short follow-up periods and small sample sizes, thus presenting a high risk of bias ,16. Puls(1)No statistical differences were observed regarding bone radiodensity after treatment with conventional healing caps vs. pulse electromagnetic stimulation caps, however, there was a difference regarding MED stimulation depending on the age of the patient, with younger patients experiencing a slightly increased degree of bone radiodensity.(2)The most important result of our study was a slightly higher radiodensity nearest the interface of dental implants, with the overall level of cancellous tissue density especially including variation in the level of trabecular bone mineralization.(3)A significant limitation of this study is the lack of a large dataset, which would be needed to offer further insight regarding pulsed electromagnetic waves and their influence on the bone. Additional research, using randomized controlled trials, should be conducted to ascertain its effectiveness compared with other treatment modalities.