Contralateral Hindlimb Research Articles

Modelling fore- and hindlimb peak vertical force differences in trotting horses using upper body kinematic asymmetry variables

Differences in peak vertical ground reaction forces (dFzpeak) between contralateral forelimbs and hindlimbs are considered the gold standard for quantifying weight-bearing lameness. However, measuring kinematics for the same purpose is more common and practical. Vertical movement asymmetries (VMA) of the horse’s upper body have previously been correlated to fore- and hindlimb lameness. But the combined response of head, withers and pelvis VMA to fore- and hindlimb dFzpeak has not yet been thoroughly investigated. Deriving the kinetic responses from kinematics would help the interpretation and understanding of quantified weight-bearing lameness.In this retrospective study, 103 horses with a wide range of fore- and hindlimb dFzpeak had been trotted on a force-measuring treadmill synchronized with an optical motion capture system. VMA of the head, withers and pelvis as well as dFzpeak were extracted. Multiple linear mixed models and linear regressions of kinematic variables were used to model the dFzpeak. It was hypothesised that all included VMA would have a significant influence on the dFzpeak outcome variables.The results showed a complex relationship between VMA and dFzpeak where both amplitude and timing of the VMA were of importance. On average, the contribution percentage of VMA to fore/hind dFzpeak were 66/34% for head, 76/24% for withers and 33/67% for pelvis. The linear regressions for the fore/hindlimb models achieved mean measurement root mean squared errors of 0.83%/0.82% dFzpeak. These results might help determine the clinical relevance of upper body VMA and distinguish between primary fore, hind, ipsilateral and diagonal weight-bearing lameness.

Hip medial rotator action of gluteus medius in Japanese macaque (Macaca fuscata) and implications to adaptive significance for quadrupedal walking in primates.

The gluteus medius (GM) muscle in quadrupedal primates has long been thought to mainly act as a hip extensor. However, previous reports argue that it may be a prime hip medial rotator and functions to rotate the pelvis in the horizontal plane, suggesting the functional differentiation between the GM and other hip extensors as hamstrings. In this study, we aim to quantify the muscle actions of the GM and hamstrings using muscle moment arm lengths and discuss the functional differentiation among hip extensors. Muscle attachment sites of eight specimens of Japanese macaque (Macaca fuscata) were digitized, and musculoskeletal models were constructed. Flexor-extensor, abductor-adductor, and medial-lateral rotator moment arms were calculated as the models were moved following the experimentally acquired kinematic data during walking on a pole substrate. Using electromyography, we also recorded the pattern of muscle activation. The GM showed a larger medial rotator moment arm length than the extensor moment arm length when it was activated, suggesting this muscle acts mainly as a hip medial rotator rather than as a hip extensor. The medial rotator action of the GM in the early support phase may rotate the pelvis in the horizontal plane and function to help contralateral forelimb reaching as a previous study suggested and facilitate contralateral hindlimb swinging to place the foot near the ipsilateral forelimb's hand.

Decoding Bilateral Hindlimb Kinematics From Cat Spinal Signals Using Three-Dimensional Convolutional Neural Network.

To date, decoding limb kinematic information mostly relies on neural signals recorded from the peripheral nerve, dorsal root ganglia (DRG), ventral roots, spinal cord gray matter, and the sensorimotor cortex. In the current study, we demonstrated that the neural signals recorded from the lateral and dorsal columns within the spinal cord have the potential to decode hindlimb kinematics during locomotion. Experiments were conducted using intact cats. The cats were trained to walk on a moving belt in a hindlimb-only condition, while their forelimbs were kept on the front body of the treadmill. The bilateral hindlimb joint angles were decoded using local field potential signals recorded using a microelectrode array implanted in the dorsal and lateral columns of both the left and right sides of the cat spinal cord. The results show that contralateral hindlimb kinematics can be decoded as accurately as ipsilateral kinematics. Interestingly, hindlimb kinematics of both legs can be accurately decoded from the lateral columns within one side of the spinal cord during hindlimb-only locomotion. The results indicated that there was no significant difference between the decoding performances obtained using neural signals recorded from the dorsal and lateral columns. The results of the time-frequency analysis show that event-related synchronization (ERS) and event-related desynchronization (ERD) patterns in all frequency bands could reveal the dynamics of the neural signals during movement. The onset and offset of the movement can be clearly identified by the ERD/ERS patterns. The results of the mutual information (MI) analysis showed that the theta frequency band contained significantly more limb kinematics information than the other frequency bands. Moreover, the theta power increased with a higher locomotion speed.

A secondary motor area contributing to interlimb coordination during visually guided locomotion inthecat.

We investigated the contribution of cytoarchitectonic cortical area 4δc, in the caudal bank of the cruciate sulcus of the cat, to the control of visually guided locomotion. To do so, we recorded the activity of 114 neurons in 4δc while cats walked on a treadmill and stepped over an obstacle that advanced toward them. A total of 84/114 (74%) cells were task-related and 68/84 (81%) of these cells showed significant modulation of their discharge frequency when the contralateral limbs were the first to step over the obstacle. These latter cells included a substantial proportion (27/68 40%) that discharged between the passage of the contralateral forelimb and the contralateral hindlimb over the obstacle, suggesting a contribution of this area to interlimb coordination. We further compared the discharge in area 4δc with the activity patterns of cells in the rostral division of the same cytoarchitectonic area (4δr), which has been suggested to be a separate functional region. Despite some differences in the patterns of activity in the 2 subdivisions, we suggest that activity in each is compatible with a contribution to interlimb coordination and that they should be considered as a single functional area that contributes to both forelimb-forelimb and forelimb-hindlimb coordination.

DEPENDENCE OF THE RESTORATIVE EFFECT OF MACROPOROUS POLY(N-[2-HYDROXYPROPYL]-METHACRYLAMIDE HYDROGEL ON THE SEVERITY OF EXPERIMENTAL LACERATIVE SPINAL CORD INJURY

restoration of the spinal cord function presents a most severe biomedical issue nowadays. The aim of the study was to detect the macroporous poly(N-[2-hydroxypropyl]-methacrylamide hydrogel (PHPMA-hydrogel, HG) restorative effect dependence on the severity of the laceration spinal cord injury in young organisms. The male rats sample (~1-month-old, ~50 g, inbred Wistar line) was represented with 4 experimental groups: 1) spinal cord lateral hemisection at the level of ~Т12–Т13 segments (Sect; n=11); 2) spinal cord lateral hemiexcision ~1 mm long at the similar level (Exc; n=8); 3) spinal cord lateral hemisection at the similar level with immediate implantation of the hydrogel fragment into the trauma region (HGsect; n=11); 4) spinal cord lateral hemiexcision at the similar level with immediate implantation of the hydrogel fragment into the affected region (HGexс; n=6). The motor function and spasticity of the paretic hindlimb was estimated respectively by the technically modified Basso–Beattie–Bresnahan (ВВВ) and Ashworth, conditionally blinded to individual characteristics of all operated animals and previous study results. The observation lasted for ~5 months. The criteria of non-inclusion were as follows: the ipsilateral hindlimb function level in a week after the injury >9 points ВВВ, and the contralateral hindlimb function level during prolonged period ≤14 points ВВВ. The results were interpreted and presented according to the standardized time scale with interpolatory representation of the motor function and spasticity individual level in certain cases. Asymptotic stage differences between the studied groups and subgroups were stated during the first three weeks as well as in 8 weeks and 3 months after the injury. We found out that in a week after injury the motor function level in group Exc made up 0.9±0.5 points ВВВ, in group HGexc — 3.6±1.2 points, in group Sect — 5.9±1.1 points, in group HGsect — 6.0±1.0 points. In 5 months the motor function level in group Sect made up 9.5±1.0 points ВВВ, in group HGsect — 9.5±1.1 points, in group Exc — 0.8±0.3 points, in group HGexc — 4.5±1.8 points. At the same study stage the spasticity level in groups Sect and HGsect was, respectively, 0.8±0.2 and 0.8±0.3 points Ashworth, in group HGexc — 1.8±0.7 points, in group Exc — 3.6±0.3 points. Throughout the study no significant differences in groups Sect and HGsect have been detected, and in groups Exc і HGexc such differences were detected only in 5 weeks after the injury. The considerable difference of spasticity in groups Sect and HGsect was noted in 1 week after the injury, in groups HGexc and Exc — during first 2 months of the experiment. In groups Sect and Exc reliable difference of both motor function and spasticity level was found at all study stages. In groups HGsect and HGexc considerable difference of the motor function level was characteristic at all stages, except for the end of the 1st and 7th weeks, whereas spasticity level differences throughout the study remained insignificant. So, the tested hydrogel in young organisms shows positive effect only with severe trauma stages accompanied with extensive spinal cord defect.

Targeting the motor cortex to restore walking after incomplete spinal cord injury.

Targeting the motor cortex to restore walking after incomplete spinal cord injury.

Model of spinal cord lateral hemi-excision at the lower thoracic level for the tasks of reconstructive and experimental neurosurgery

Purpose. To test the model of spinal cord lateral hemiexcision in young rats. Materials and methods. Animals ‒ male rats (age about 1 month, body weight about 50 g, inbred derivatives of the Wistar line); the number of experimental groups is: 1) lateral spinal cord hemisection at the level of segments about T12–T13 (Sect; n=11); 2) lateral spinal cord hemiexcision about 1 mm long at the similar level (Exc; n=8). Assessment of motor Function Index (FI) and the Spasticity Index (SI) of the paretic hindlimb was carried out using the Basso–Beattie–Bresnahan (BBB) scale and Ashworth scale, respectively, in our technical modifications. The non-inclusion criteria: the BBB score above 9 points of FI for the ipsilateral hindlimb in a week after injury ‒and / or BBB score less than or equal to 14 points of FI of the contralateral hindlimb during a long follow-up period (in general, 2 animals in the Sect group, 3 animals ‒ in the Exc group). Asymptotic differences in the timing of testing between subgroups and groups were revealed during the first three weeks of follow-up. Interpolation reproduction of individual values of FI and SI was used in exceptional cases. The total follow-up period was 5 months. Statistical analysis was performed using the Mann-Whitney U Test, Wilcoxon Matched Pairs Test, Spearman’s Rank Order Correlation. For pathomorphological study, the method of silver impregnation of the spinal cord longitudinal sections of the Exc group animals obtained in 5 months after the simulation of injury was used. Results. One week after injury, the FI in the Sect group was 5.9±1.1 according to BBB points, a statistically significant increase in the FI lasted for the first 3 weeks (p<0.05; Wilcoxon Matched Pairs Test), the FI maximum in the group was 10.1±1.1 BBB points, and the FI value at the end of the study was 9.5±1.0 BBB points. In the Exc group, 1 week after injury, the FI was 0.9±0.5 BBB points, during the next week it reached the actual maximum (1.9±0.7 BBB points), by the end of the 5th month it significantly decreased to 0.8±0.3 BBB points (p<0.05; Wilcoxon Matched Pairs Test). One week after injury, the SI value in the Sect group was 0.3±0.1 points according to Ashworth scale, in the Exc group ‒ 0.7±0.1 Ashworth points, a significant increase (p<0.05; Wilcoxon Matched Pairs Test) in SI in the Sect group was noted during the 2nd week and the 2nd month, in the Exc group ‒ during the 2nd and 6th week, as well as the 3rd and 5th month after injury. The SI final and maximal score for the Sect group was 0.8±0.2 Ashworth points, and for the Exc group ‒ 3.6±0.3 Ashworth points. For both groups, there was no correlation between the mean FI value and a significant positive correlation of the mean SI value with the value of the follow-up period (p<0.05; Spearman’s Rank Order Correlation), as well as the absence of correlation between the mean FI and SI values during the total follow-up period. A significant negative correlation (p<0.05; Spearman’s Rank Order Correlation) between individual FI and SI values was found after 1 and 4 weeks, 3 and 5 months after the injury for the Sect group, as well as after 5, 7, 8 weeks and after 3 and 4 months for the Exc group. At all periods of follow-up, the difference in both FI and SI mean values of both groups was significant (p<0.05; the Mann-Whitney U Test). Conclusions. The studied model of spinal cord injury in young rats is the means of choice for testing solid neural transplantation means for the spinal cord injury restorative treatment. The interpretation of data obtained using the BBB scale on models of lateral half spinal cord injury should be carried out with caution, and the methodology for verifying spasticity requires significant improvement. We recommend that the optimal timing for the FI and SI monitoring after lateral half spinal cord injury is 7 days, 14 days and in 1, 2, 3, 4, 5, 6, and 7 months.

Lactate administration does not affect denervation-induced loss of mitochondrial content and muscle mass in mice.

Lactate is considered to be a signaling molecule that induces mitochondrial adaptation and muscle hypertrophy. The purpose of this study was to examine whether lactate administration attenuates denervation‐induced loss of mitochondrial content and muscle mass. Eight‐week‐old male Institute of Cancer Research mice underwent unilateral sciatic nerve transection surgery. The contralateral hindlimb served as a sham‐operated control. From the day of surgery, mice were injected intraperitoneally with PBS or sodium lactate (equivalent to 1 g·kg−1 body weight) once daily for 9 days. After 10 days of denervation, gastrocnemius muscle weight decreased to a similar extent in both the PBS‐ and lactate‐injected groups. Denervation significantly decreased mitochondrial enzyme activity, protein content, and MCT4 protein content in the gastrocnemius muscle. However, lactate administration did not have any significant effects. The current observations suggest that daily lactate administration for 9 days does not affect denervation‐induced loss of mitochondrial content and muscle mass.

Deleterious Effects of Remote Ischaemic Per-conditioning During Lower Limb Ischaemia–Reperfusion in Mice

The aim of this study was to investigate whether remote ischaemic per-conditioning might protect skeletal muscle during lower limb ischaemia-reperfusion (IR). Twenty-three male C57BL/6 mice were randomised into three groups: sham group (n= 7), IR group (unilateral tourniquet induced three hours of ischaemia followed by 24 hours of reperfusion, n= 8), and remote ischaemic per-conditioning group (RIPerC) (three cycles of 10 minute IR episodes on the non-ischaemic contralateral hindlimb, n= 8). Oxygraphy, spectrofluorometry, and electron paramagnetic resonance spectroscopy were performed in order to determine mitochondrial respiratory chain complexes activities, mitochondrial calcium retention capacity (CRC) and reactive oxygen species (ROS) production in skeletal muscle. IR impaired mitochondrial respiration (3.66 ± 0.98 vs. 7.31 ± 0. 54 μmol/min/g in ischaemic and sham muscles, p= .009 and p= .003 respectively) and tended to impair CRC (2.53 ± 0.32 vs. 3.64 ± 0.66 μmol/mg in ischaemic and sham muscles respectively, p= .066). IR did not modify ROS production (0.082 ± 0.004 vs. 0.070 ± 0.004 μmol/min/mg in ischaemic and sham muscles respectively, p= .74). RIPerC failed to restore mitochondrial respiration (3.82 ± 0.40 vs. 3.66 ± 0.98 μmol/min/g in ischaemic muscles from the RIPerC group and the IR group respectively, p= .45) and CRC (2.76 ± 0.3 vs. 2.53 ± 0.32 μmol/mg in ischaemic muscles from the RIPerC group and the IR group respectively, p= .25). RIPerC even impaired contralateral limb mitochondrial respiration (3.85 ± 0.34 vs. 7.31 ± 0. 54 μmol/min/g in contralateral muscles and sham muscles respectively, -47.3%, p= .009). RIPerC failed to protect ischaemic muscles and induced deleterious effects on the contralateral non-ischaemic muscles. These data do not support the concept of RIPerC.

Microstimulation of the Premotor Cortex of the Cat Produces Phase-Dependent Changes in Locomotor Activity.

To determine the functional organization of premotor areas in the cat pericruciate cortex we applied intracortical microstimulation (ICMS) within multiple cytoarchitectonically identified subregions of areas 4 and 6 in the awake cat, both at rest and during treadmill walking. ICMS in most premotor areas evoked clear twitch responses in the limbs and/or head at rest. During locomotion, these same areas produced phase-dependent modifications of muscle activity. ICMS in the primary motor cortex (area 4γ) produced large phase-dependent responses, mostly restricted to the contralateral forelimb or hindlimb. Stimulation in premotor areas also produced phase-dependent responses that, in some cases, were as large as those evoked from area 4γ. However, responses from premotor areas had more widespread effects on multiple limbs, including the ipsilateral limbs, than did stimulation in 4γ. During locomotion, responses in both forelimb and hindlimb muscles were evoked from cytoarchitectonic areas 4γ, 4δ, 6aα, and 6aγ. However, the prevalence of effects in a given limb varied from one area to another. The results suggest that premotor areas may contribute to the production, modification, and coordination of activity in the limbs during locomotion and may be particularly pertinent during modifications of gait.

Nerve gaps repaired with acellular nerve allografts recellularized with Schwann-like cells: Preclinical trial

Acellular nerve allografts (ANA) recellularized with mesenchymal stem cells (MSC) or Schwann cells (SC) are, at present, a therapeutic option for peripheral nerve injuries (PNI). This study aimed to evaluate the regenerative and functional capacity of a recellularized allograft (RA) compared with autograft nerve reconstruction in PNI. Fourteen ovines were randomly included in two groups (n=7). A peroneal nerve gap 30 mm in length was excised, and nerve repair was performed by the transplantation of either an autograft or a recellularized allograft with SC-like cells. Evaluations included a histomorphological analysis of the ANA, MSC pre differentiated into SC-like cells, at one year follow-up functional limb recovery (support and gait), and nerve regeneration using neurophysiological tests and histomorphometric analysis. All evaluations were compared with the contralateral hindlimb as the control. The nerve allograft was successfully decellularized and more than 70% of MSC were pre differentiated into SC-like cells. Functional assessment in both treated groups improved similarly over time (p <0.05). Neurophysiological results (latency, amplitude, and conduction velocity) also improved in both treated groups at twelve months. Histological results demonstrated a less organized arrangement of nerve fibers (p <0.05) with an active remyelination process (p <0.05) in both treated groups compared with controls at twelve months. ANA recellularized with SC-like cells proved to be a successful treatment for nerve gaps. Motor recovery and nerve regeneration were satisfactorily achieved in both graft groups compared with their contralateral nontreated nerves. This approach could be useful for the clinical therapy of PNI.

Distinct patterns of activity in individual cortical neurons and local networks in primary somatosensory cortex of mice evoked by square-wave mechanical limb stimulation.

Artificial forms of mechanical limb stimulation are used within multiple fields of study to determine the level of cortical excitability and to map the trajectory of neuronal recovery from cortical damage or disease. Square-wave mechanical or electrical stimuli are often used in these studies, but a characterization of sensory-evoked response properties to square-waves with distinct fundamental frequencies but overlapping harmonics has not been performed. To distinguish between somatic stimuli, the primary somatosensory cortex must be able to represent distinct stimuli with unique patterns of activity, even if they have overlapping features. Thus, mechanical square-wave stimulation was used in conjunction with regional and cellular imaging to examine regional and cellular response properties evoked by different frequencies of stimulation. Flavoprotein autofluorescence imaging was used to map the somatosensory cortex of anaesthetized C57BL/6 mice, and in vivo two-photon Ca2+ imaging was used to define patterns of neuronal activation during mechanical square-wave stimulation of the contralateral forelimb or hindlimb at various frequencies (3, 10, 100, 200, and 300 Hz). The data revealed that neurons within the limb associated somatosensory cortex responding to various frequencies of square-wave stimuli exhibit stimulus-specific patterns of activity. Subsets of neurons were found to have sensory-evoked activity that is either primarily responsive to single stimulus frequencies or broadly responsive to multiple frequencies of limb stimulation. High frequency stimuli were shown to elicit more population activity, with a greater percentage of the population responding and greater percentage of cells with high amplitude responses. Stimulus-evoked cell-cell correlations within these neuronal networks varied as a function of frequency of stimulation, such that each stimulus elicited a distinct pattern that was more consistent across multiple trials of the same stimulus compared to trials at different frequencies of stimulation. The variation in cortical response to different square-wave stimuli can thus be represented by the population pattern of supra-threshold Ca2+ transients, the magnitude and temporal properties of the evoked activity, and the structure of the stimulus-evoked correlation between neurons.

Mechanical sensitization, increased axonal excitability, and spontaneous activity in C-nociceptors after ultraviolet B irradiation in pig skin.

Ultraviolet B (UVB) irradiation induces hyperalgesia in human and animal pain models. We investigated mechanical sensitization, increase in axonal excitability, and spontaneous activity in different C-nociceptor classes after UVB in pig skin. We focused on units with receptive fields covering both irradiated and nonirradiated skin allowing intraindividual comparisons. Thirty-five pigs were irradiated in a chessboard pattern, and extracellular single-fibre recordings were obtained 10 to 28 hours later (152 fibers). Units from the contralateral hind limb served as a control (n = 112). Irradiated and nonirradiated parts of the same innervation territory were compared in 36 neurons; low threshold C-touch fibers (n = 10) and sympathetic efferents (n = 2) were unchanged, but lower mechanical thresholds and higher discharge frequency at threshold were found in mechanosensitive nociceptors (n = 12). Half of them could be activated with nonnoxious brush stimuli in the sunburn. Four of 12 mechanoinsensitive nociceptors were found sensitized to mechanical stimulation in the irradiated part of the receptive field. Activity-dependent slowing of conduction was reduced in the irradiated and in the nonirradiated skin as compared with the control leg, whereas increased ability to follow high stimulation frequencies was restricted to the sunburn (108.5 ± 37 Hz UVB vs 6.3 ± 1 Hz control). Spontaneous activity was more frequent in the sunburn (72/152 vs 31/112). Mechanical sensitization of primary nociceptors and higher maximum after frequency are suggested to contribute to primary hyperalgesia, whereas the spontaneous activity of silent nociceptors might offer a mechanistic link contributing to ongoing pain and facilitated induction of spinal sensitization.

Upper body movement analysis of multiple limb asymmetry in 367 clinically lame horses

Compensatory lameness is common in horses and evaluation can be challenging. To investigate patterns of compensatory movements in clinical cases with fore- or hindlimb lameness before and after diagnostic analgesia. Retrospective clinical study. Multiple limb lameness of 367 horses was characterised by type (push-off, impact or mixed), limb (fore- or hindlimb in predominant lameness) and side (ipsi- or contralateral in concurrent lameness) using a body-mounted inertial sensor (BMIS). Diagnostic analgesia was performed until the percentage improvement of the vector sum in forelimb lameness and the mean difference of the maximum or minimum pelvic height (PDmax or PDmin ) in hindlimb lameness was ≥50%. Linear mixed model and post-estimation of effects were performed by contrast command with multiple comparisons adjusted by Bonferroni method. Correlation of pre- and post-analgesia of all head and pelvis asymmetry parameters was tested with Spearman's rank correlation. Improvement in vector sum per mm after diagnostic analgesia in forelimb impact lameness positively correlated with decrease in PDmax in contralateral mixed lameness (0.187mm, r=.58, P<.05). Improvement in PDmin per mm after diagnostic analgesia in hindlimb mixed and PDmax in hindlimb push-off lameness decreased vector sum in ipsilateral forelimb impact lameness by 0.570 and 0.696mm, respectively (P<.05), with no positive correlation. A variety of cases with inhomogeneous distribution of lameness patterns was investigated retrospectively, therefore, it is impossible to distinguish between true multiple limb lameness and compensatory lameness in this clinical material. Various asymmetry patterns of concurrent lameness were seen in horses with naturally occurring primary forelimb impact lameness with contralateral compensatory hindlimb lameness with a mixed component being the most common. In horses with hindlimb lameness, compensatory movements were seen in ipsilateral forelimbs, mostly as an ipsilateral impact lameness during straight line trot.

Riding Soundness—Comparison of Subjective With Objective Lameness Assessments of Owner-Sound Horses at Trot on a Treadmill

Lameness is a symptom indicative of pain or injury of the locomotor apparatus. Lame horses generally should not be ridden. However, owners’ ability to assess lameness has been questioned. This study’s aim was to use subjective lameness assessments and objective gait analysis to generate a descriptive overview of movement and weight-bearing asymmetries of owner-sound riding horses. 235 horses were subjectively assessed in a field study, and the owner’s perception of their horse’s orthopedic health was recorded through an online survey. 69 horses were re-evaluated by gait analysis at an equine hospital. During trot on an instrumented treadmill, the gait was scored by a veterinarian using lameness grades from 0/5 (sound) to 3/5 (moderate lameness visible at trot). Movement asymmetry of the head (HDmin) and pelvis (PDmin) and weight-bearing asymmetry were quantified simultaneously. The prevalence of subjectively scored lameness grade ≥2/5 in one or more limbs was 55% during study part 1 and 74% during study part 2. Movement asymmetry of the head and/or pelvis exceeding HDmin ≥12 mm and/or PDmin ≥6 mm was found in 57% of the horses. 58% showed weight-bearing asymmetries between contralateral front and/or hind limbs of ≥3% body mass. Gait analysis showed considerable variability of movement and weight-bearing asymmetry values, sometimes independent of the clinical lameness grade, especially in the forehand. Several horses with lameness grade ≤1/5 had asymmetry values greater than mentioned thresholds. The analysis of movement and weight-bearing asymmetry revealed that these objective variables did not necessarily act uniformly and therefore should be interpreted with caution.

Skin temperature contribution to the decrease in withdrawal latency following chronic constriction injury

BackgroundChronic constriction injury (CCI) is widely used as an animal neuropathic pain model. Neuropathic pain is considered to exist when withdrawal latency to thermal stimulation is decreased after inducing a CCI to the sciatic nerve. However, it is known that CCI leads to changes in skin temperature and that skin temperature can affect withdrawal latency. Aim of this study was to compare withdrawal latencies of constricted and contralateral hind limbs, to thermal stimulation, at the same artificially-induced skin temperatures. MethodsNeuropathic pain was induced by four ligatures on the left sciatic nerve in adult male Wistar rats. Withdrawal latencies were measured from the 11th to 14th day after ligation, in different ambient temperatures, using the plantar test (Hargreaves method). By changing ambient we produced different hind limb skin temperatures. ResultsOur results show that (1) CCI cause an increase in skin temperature; (2) the withdrawal latency was inversely related to ambient and skin temperature in the same manner for both the ligated and contralateral hind limbs; and (3) withdrawal latencies did not differ significantly for the ligated and contralateral hind limbs when the temperature of the hind limbs was artificially made the same (i.e., by changing the ambient temperature). ConclusionsWithdrawal latencies to thermal stimulation did not differ on ligated and contralateral hind limb after CCI to the sciatic nerve if the temperature of the hind limbs was artificially or mathematically made the same. This finding may have significant impact on the interpretation results of neuropathic pain research.

Bioinformatics Analysis of the Molecular Mechanism of Late-Stage Heterotopic Ossification.

Background Heterotopic ossification (HO) is a common disease happened in soft tissues after injury. The present study utilized the bioinformatics method to analyze the HO samples in a mouse burn/tenotomy-induced HO model to identify the possible key points and treatment targets. Methods The transcriptome profiles of GSE126118 were obtained from the Gene Expression Omnibus (GEO) database. The study was based on a mouse burn/tenotomy-induced HO model, and 2 tenotomy samples and 3 uninjured contralateral hindlimb tendon samples were collected at 3 weeks after injury for further analysis. The transcripts per million approach was performed for background correction and normalization; then, the differentially expressed genes (DEGs) were detected using the limma R package with the settings p < 0.01 and ∣log2FC∣ > 2.0. The Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, and the protein-protein interaction (PPI) network analysis were performed with the detected DEGs. Results A total of 74 DEGs were upregulated, and 159 DEGs were downregulated between the tenotomy and uninjured tendon group. Pathway and process enrichment analyses demonstrated that the upregulated DEGs were mainly associated with terms related to ECM remodeling, ossification, angiogenesis, inflammation, etc., and the downregulated DEGs were mainly associated with oxidative phosphorylation, metabolic process, etc. Conclusion The results of GO, KEGG, and PPI network analyses suggested that the ECM remodeling, ossification, angiogenesis, and inflammation processes were markedly upregulated in the tenotomy site. And the oxidative phosphorylation and metabolic processes were markedly downregulated. These findings provide valuable clues for highlighting the characteristics of late-stage HO and investigating possible treatments.

Abstract 43: Evaluation The Effect Of Brain-death Physiology And Immunosuppression On Muscle-derived Stem Cell Expansion: Study On Porcine Tissue

Background: Muscle-derived stem cells (MDSCs) are robust mediators of tissue regeneration that have been shown, in the literature, to exhibit multipotency in murine models. We hypothesize that immunosuppression, transplant physiology, and braindeath negatively affect the stem cell-niche in vascularized composite allografts (VCA) and may affect tolerance/rejection. Our study aim was to evaluate the growth of MDSCs under these conditions in a porcine VCA model. Methods: Muscle samples were harvested from the hind-limbs of pig before and after inducing brain-death. Samples were then harvested from both the VCA of another immunosuppressed pig and from the contralateral hindlimb of the same recipient 7 days after transplantation. MDSC populations were isolated using a modified collagen-sorting preplate technique. Imaging was obtained at various time points (t=0-10 days). Images were analyzed for confluence using ImageJ, statistical analysis was performed using Mathematica. Results: Time to confluence (t = 3, 5, 7, 10 days) between pre-braindeath and post-braindeath MDSC populations was significantly different (20%, 38%, 75%, 81% vs. 7%, 17%, 31%, 39%) (p<0.01). Time to confluence (t = 2, 4, 6, 8, 10 days) between immunosuppressed recipient and allograft MDSC populations was significantly different (3%, 6%, 13%, 45%, 55% vs. 7% 11%, 39%, 53%, 71%, respectively) (p=0.02). At t=10 days, mean confluence between pre-BD and immunosuppressed recipient MDSC populations was significantly different (81% vs. 55%) (p < 0.01); similarly, mean confluence between pre-braindeath and allograft MDSC populations was also significantly different (81% vs. 71%) (p = 0.01). At t=10 days, mean confluence between post-braindeath and immunosuppressed recipient MDSC populations was significantly different (39% vs. 55%) (p<0.01). Conclusion: Brain-death and immunosuppression significantly impair MDSC expansion and mitosis based on time to confluence; however, transplantation may ameliorate the negative effects of immunosuppression by acting as a surrogate for trauma and inflammation. Brain-death physiology appears to have the greatest impact on MDSC expansion in our study. Further studies will focus on immune modulation and scaffold growth in porcine MDSC populations.

Novel Contributions of Neutrophil‐derived Myeloperoxidase and Hypochlorous Acid to 20‐HETE Production that drives Post‐Ischemic Angiogenesis

Objective20‐hydroxy‐eicosatetraenoic acid (20‐HETE), an arachidonic acid metabolite produced by cytochrome P450 (CYP) 4A/F ω‐hydroxylases, was recently uncovered as a novel contributor to ischemia‐induced angiogenesis. The current study aims to determine whether neutrophil‐derived myeloperoxidase (MPO) and hypochlorous acid (HOCl) mediate post‐ischemic 20‐HETE increases that drive angiogenesis and its underlying mechanism.MethodsMice were subjected to a femoral artery ligation with the contralateral hindlimb acting as the non‐ischemic control. Hindlimb angiogenesis was assessed by laser doppler blood perfusion imaging (LDPI) as well as quantifying the microvascular density in the gracilis muscles where angiogenesis is taking place. Endothelial cells (EC) in culture were subjected to physiological concentrations of MPO and HOCl. Gracilis muscles and EC were analyzed for 20‐HETE production by LC/MS/MS. The mRNA expression of the 20‐HETE synthesizing enzyme, CYP4A11 was measured by real‐time PCR. Protein expression of CYP4A11 and HIF1a were analyzed using western blotting. The rate and mechanism of HOCl entry into EC was evaluated using live‐cell fluorescence microscopy with a HOCl‐specific indicator dye (FDOCl‐1) in the presence/absence of chloride channel blocker 9‐anthracenecarboxylic acid (9‐Ac) and/or gap junction inhibitor 18‐beta‐glycyrrhetinic acid (18β‐GA).ResultsIn animals that were selectively depleted of neutrophils, ischemia failed to induce 20‐HETE increases in the gracilis muscle compared to the controls (13 ± 1.5 vs 35 ± 5 pg/mg of protein). Additionally, MPO‐null mice failed to show increased post‐ischemic 20‐HETE (~2 ± .25 vs 35 ± 5 pg/mg of protein) as well. Both animals display a decreased post‐ischemic angiogenenic phenotype. In EC, the addition of MPO or HOCl markedly stimulated the expression of CYP4A11 and production of 20‐HETE (40 ± 12 vs 8 ± 5 pg/mg of protein). Upregulation of CYP4A11 transcript/protein (&gt;2.5‐fold) and HIF1a protein expression (2‐fold) was observed as early as 15 minutes post‐HOCl exposure. The mechanism of HOCl entry was also exmained by FDOCl‐1 fluorescence quantification, which was markedly decreased in EC pre‐treated with 9‐Ac or 18β‐GA by approximately 40% and 60%, respectively.ConclusionOur study demonstrates for the first time the contribution of MPO and HOCl in promoting 20‐HETE production that drives post‐ischemic angiogenesis. Moreover, this study aims to further expand on the known mechanisms involved in regulating ischemia‐induced angiogenesis. These new findings have high clinical relevancy in studying vascular pathologies by identifing novel mechanisms that contribute to pathological angiogenesis.Support or Funding InformationThis study was supported by grants from AHA[11SDG6870004 (AMG) and 17GRNT33430003 (AMG)], the NIH [HL34300 (M.L.S.) and grant DK38226 (J.R.F.)]; &amp; the R.A. Welch Foundation [GL625910 (J.R.F.)].

Abstract 71: Microsurgical Induced Angiogenesis

Purpose: Blood vessel growth is essential for the viability of all tissues. Angiogenesis is paramount to normal development, injury recovery, and more recently, vital to the integration of bioengineered grafts. However, innate blood vessel proliferation is slow and often inadequate. Therapeutic angiogenesis aims to stimulate the growth of new blood vessels from preexisting vasculature using various strategies such as exogenous pharmacologic stimulation, cytokine supplementation, stem cells, and in situ cell homing. However, widespread clinical translation has proven suboptimal and the side-effects significant. Here we describe a novel microsurgical intervention to safely induce rapid therapeutic angiogenesis, which could be used in a variety of settings. Methods: The rat hindlimb macrovasculature (N=10) was micropunctured (MP) using a 60 μm diameter needle at defined intervals prior to implantation of a prepared Type I collagen scaffold directly on top of the punctured segment. The contralateral hindlimb served as a normal (non-micropunctured) intrinsic control. Collagen grafts and the underlying vascular segment were explanted en bloc for gross and histologic analysis after 24, 48, and 72 hours. Cellular infiltration and capillary ingrowth were assessed per high powered field (HPF) following trichrome staining. Single cell-lined lumens were characterized by both number and diameter; from which the overall perfusable area was calculated. The presence of endothelial cells was verified by CD31/PECAM-1, von Willebrand factor (vWF), and Tie2 immunofluorescence staining. Endothelial cell proliferation was estimated by VEGFR2, Notch 1, and delta-like 4 (DLL4). Statistical significance was determined using the one-tailed Mann-Whitney U test. Results: Micropunctured vessels demonstrated an increase in cellular infiltration, capillary outgrowth and luminal diameter across all time points when compared to non-micropunctured samples. This resulted in a two-fold overall increase of perfusable area within the explanted collagen, reaching statistical significance (P<0.05) at 24 and 72 hours. Immunofluorescence labeling of CD31, vWF, and Tie2 confirmed the increased presence of endothelial cells along the periphery of measured luminal structures in micropunctured specimens. Additionally, micropunctured samples exhibited increased cellular staining of VEGFR2, Notch 1, and DLL4. This suggests the specific proliferation of endothelial stalk and tip cells which are fundamental to sprouting angiogenesis. Conclusion: Microsurgical induction of angiogenesis was first described with the arduous creation of arteriovenous-loops. However, the time to new capillary formation is slow, limiting widespread clinical translation. Here we describe an innovative alternative technique that leads to the rapid proliferation of endothelial cells and robust capillary formation. Because our approach evaluates the paradigm of therapeutic angiogenesis from a completely novel perspective it has enormous potential to mitigate ischemia in both conventional surgery and bioengineered applications.