Intact Hindlimb Research Articles

Effect of Ultrasound Pulse Length on Sonoreperfusion Therapy.

In recent years, long- and short-pulse ultrasound (US)-targeted microbubble cavitation (UTMC) has been found to increase perfusion in healthy and ischemic skeletal muscle, in pre-clinical animal models of microvascular obstruction and in the myocardium of patients presenting with acute myocardial infarction. There is evidence that the observed microvascular vasodilation is driven by the nitric oxide pathway and purinergic signaling, but the time course of the response and the dependency on US pulse length are not well elucidated. Because our prior data supported that sonoreperfusion efficacy is enhanced by long-pulse US versus short-pulse US, in this study, we sought to compare long-pulse (5000 cycles) and short-pulse (500×10 cycles) US at a pressure of 1.5 MPa with an equivalent total number of acoustical cycles, hence constant acoustic energy, and at the same frequency (1 MHz), in a rodent hind limb model with and without microvascular obstruction (MVO). In quantifying perfusion using burst replenishment contrast-enhanced US imaging, we made three findings: (i) Long and short pulses result in different vasodilation kinetics in an intact hind limb model. The long pulse causes an initial spasmic reduction in flow that spontaneously resolved at 4 min, followed by sustained higher flow rates (approximately twofold) compared with baseline, starting 10 min after therapy (p < 0.05). The short pulse caused a short-lived approximately twofold increase in flow rate that peaked at 4 min (p < 0.05), but without the initial spasm. (ii) The sustained increased response with the long pulse is not simply reactive hyperemia. (iii) Both pulses are effective in reperfusion of MVO in our hindlimb model by restoring blood volume, but only the long pulse caused an increase in flow rate after treatment ii, compared with MVO (p < 0.05). Histological analysis of hind limb muscle post-UTMC with either pulse configuration indicates no evidence of tissue damage or hemorrhage. Our findings indicate that the microbubble oscillation induces vasodilation, and therapeutic efficacy for the treatment of MVO can be tuned by varying pulse length; relative to short-pulse US, longer pulses drive greater microbubble cavitation and more rapid microvascular flow rate restoration after MVO, warranting further optimization of the pulse length for sonoreperfusion therapy.

Electrical Stimulation of Distal Tibial Nerve During Stance Phase of Walking May Reverse Effects of Unilateral Paw Pad Anesthesia in the Cat.

Cutaneous feedback from feet is involved in regulation of muscle activity during locomotion, and the lack of this feedback results in motor deficits. We tested the hypothesis that locomotor changes caused by local unilateral anesthesia of paw pads in the cat could be reduced/reversed by electrical stimulation of cutaneous and proprioceptive afferents in the distal tibial nerve during stance. Several split-belt conditions were investigated in four adult female cats. In addition, we investigated the effects of similar distal tibial nerve stimulation on overground walking of one male cat that had a transtibial, bone-anchored prosthesis for 29months and, thus, had no cutaneous/proprioceptive feedback from the foot. In all treadmill conditions, cats walked with intact cutaneous feedback (control), with right fore- and hindpaw pads anesthetized by lidocaine injections, and with a combination of anesthesia and electrical stimulation of the ipsilateral distal tibial nerve during the stance phase at 1.2× threshold of afferent activation. Electrical stimulation of the distal tibial nerve during the stance phase of walking with anesthetized ipsilateral paw pads reversed or significantly reduced the effects of paw pad anesthesia on several kinematic variables, including lateral center of mass shift, cycle and swing durations, and duty factor. We also found that stimulation of the residual distal tibial nerve in the prosthetic hindlimb often had different effects on kinematics compared with stimulation of the intact hindlimb with paw anesthetized. We suggest that stimulation of cutaneous and proprioceptive afferents in the distal tibial nerve provides functionally meaningful motion-dependent sensory feedback, and stimulation responses depend on limb conditions.

Abstract 14673: The Effect of Increasing the Ultrasound Pulse Length on Sonoreperfusion Therapy

Background: In recent years, long and short pulse ultrasound (US)-targeted microbubble cavitation (UTMC) has been shown to increase perfusion in healthy and ischemic skeletal muscle, in pre-clinical animal models of microvascular obstruction, and in the myocardium of patients presenting with acute myocardial infarction, and microvascular vasodilation played an important role in its efficacy. Our prior preliminary data suggested that long pulse US confers enhanced sonoreperfusion efficacy compared to short pulse US. Objective: In this study, we sought to definitively compare acoustic energy matched, long (5000 cycles) and short pulse (500 х 10 cycles, separated by 100 μs intervals) US (pressure of 1.5 MPa with an equivalent total number of acoustical cycles and at the same frequency (1 MHz)), in a rodent hindlimb model, with and without microvascular obstruction (MVO). Results: By quantifying perfusion using burst replenishment contrast enhanced US imaging, we found that: (1) long and short pulses result in different vasodilation kinetics in an intact hindlimb model. The long pulse causes an initial reduction in flow due to microvascular spasm that spontaneously resolved at 4-min, followed by sustained higher flow rates (~2-folds) compared to baseline, starting 10 min after therapy ( p &lt;0.05). The short pulse caused a short-lived ~2-fold increase in flow rate that peaked at 4-min ( p &lt;0.05), but without the initial microvascular spasm; (2) the sustained increase in perfusion during long pulse is not simply reactive hyperemia; and (3) both pulses are effective in reperfusion of MVO in our hindlimb model by restoring blood volume, but only the long pulse caused an increase in flow rate after treatment 2, compared to MVO ( p &lt;0.05). Histological analysis post UTMC with either pulse configuration indicates no evidence of tissue damage or hemorrhage. Conclusions: Our findings demonstrate that the microbubble oscillation induces vasodilation and therapeutic efficacy for the treatment of MVO can be tuned by varying pulse length; relative to short pulse US, longer pulses drive greater microbubble cavitation and more rapid microvascular flow rate restoration after MVO, warranting further optimization of the pulse length for sonoreperfusion therapy.

Kinematic patterns for hindlimb obstacle avoidance during sheep locomotion

ABSTRACTFunctional recovery following general nerve reconstruction is often associated with poor results. Comparing to rat and mice experimental studies, there are much fewer investigations on nerve regeneration and repair in the sheep, and there are no studies on this subject using gait analysis in the sheep model as an assessment tool. Additionally, this is the first study evaluating obstacle negotiation and the compensatory strategies that take place at each joint in response to the obstacle during locomotion in the sheep model. This study aims to get kinematic data to serve as a template for an objective assessment of the ankle joint motion in future studies of common peroneal nerve (CP) injury and repair in the ovine model. Our results show that a moderately high obstacle set to 10% of the sheep’s hindlimb length was associated to several spatial and temporal strategies in order to increase hoof height during obstacle negotiating. Sheep efficiently cleared an obstacle by increasing knee, ankle and metatarsophalangeal flexion during swing, whereas the hip joint is not affected. This study establishes the bounds of normal motion in the neurologically intact hindlimb when approached and cleared an obstacle and provides baseline data for further studies of peripheral nerve research in the ovine model.

Contralateral peripheral neurotization for a hemiplegic hindlimb after central neurological injury.

OBJECTIVE Contralateral peripheral neurotization surgery has been successfully applied to rescue motor function of the hemiplegic upper extremity in patients with central neurological injury (CNI). It may contribute to strengthened neural pathways between the contralesional cortex and paretic limbs. However, the effect of this surgery in the lower extremities remains unknown. In the present study the authors explored the effectiveness and safety of contralateral peripheral neurotization in treating a hemiplegic lower extremity following CNI in adult rats. METHODS Controlled cortical impact (CCI) was performed on the hindlimb motor cortex of 36 adult Sprague-Dawley rats to create severe unilateral traumatic brain injury models. These CCI rats were randomly divided into 3 groups. At 1 month post-CCI, the experimental group (Group 1, 12 rats) underwent contralateral L-6 to L-6 transfer, 1 control group (Group 2, 12 rats) underwent bilateral L-6 nerve transection, and another control group (Group 3, 12 rats) underwent an L-6 laminectomy without injuring the L-6 nerves. Bilateral L-6 nerve transection rats without CCI (Group 4, 12 rats) and naïve rats (Group 5, 12 rats) were used as 2 additional control groups. Beam and ladder rung walking tests and CatWalk gait analysis were performed in each rat at baseline and at 0.5, 1, 2, 4, 6, 8, and 10 months to detect the skilled walking functions and gait parameters of both hindlimbs. Histological and electromyography studies were used at the final followup to verify establishment of the traumatic brain injury model and regeneration of the L6-L6 neural pathway. RESULTS In behavioral tests, comparable motor injury in the paretic hindlimbs was observed after CCI in Groups 1-3. Group 1 started to show significantly lower slip and error rates in the beam and ladder rung walking tests than Groups 2 and 3 at 6 months post-CCI (p < 0.05). In the CatWalk analysis, Group 1 also showed a higher mean intensity and swing speed after 8 months post-CCI and a longer stride length after 6 months post-CCI than Groups 2 and 3 (p < 0.05). Transection of L-6 resulted in transient skilled walking impairment in the intact hindlimbs in Groups 1 and 2 (compared with Group 3) and in the bilateral hindlimbs in Group 4 (compared with Group 5). All recovered to baseline level within 2 months. Histological study of the rat brains verified comparable injured volumes among Groups 1-3 at final examinations, and electromyography and toluidine blue staining indicated successful regeneration of the L6-L6 neural pathways in Group 1. CONCLUSIONS Contralateral L-6 neurotization could be a promising and safe surgical approach for improving motor recovery of the hemiplegic hindlimb after unilateral CNI in adult rats. Further investigations are needed before extrapolating the present conclusions to humans.

Comparative Analysis of Force Reactions of Rat Hindlimb Muscles after Unilateral Compression of the Sciatic Nerve and Systemic Injections of Gamma-Hydroxybutyrate

We compared force activity of the hindlimb muscles (ankle flexors and extensors and toe flexors) in rats subjected to course systemic injections of sodium gamma-hydroxybutyrate, GHOB (100 mg/kg, i.p., daily, during 4 weeks) after unilateral compression of the sciatic nerve (SN). Late effects of GHOB injections (10th and 12th weeks after surgery) were also analyzed. The contractile force of muscles was estimated using a tensometric technique. In the control group, animals were subjected to compression of the SN but were not injected with GHOB. In the experimental group, an appreciable functional loss (20-24%) was observed in muscles on the side of SN compression a week after surgery, while on the contralateral side we found a functional increment (60-17%). Within the 2nd to the 4th week of GHOB injections, stable force increments in both extensors and flexors (respectively, 2 and 2.5 times and by 20-50%) were manifested, while increments of the contractile force of muscles of the contralateral intact hindlimb (30-40%) did not depend on the function of the muscles. Within late time intervals (10th and 12th weeks) after surgery, the contractile force of muscles of the injured and contralateral hindlimbs increased maximally by 30-40%, as compared with to the control; this increment in the injured hindlimb was observed in extensors, while it was manifested in flexors of the contralateral hindlimb.

Effects of Systemic Injections of Gamma-Hydroxybutyrate on the Recovery of Functions of the Distal Hindlimb Muscles in Rats after Compression of the Sciatic Nerve

We studied the effects of systemic injections of sodium gamma-hydroxybutyrate (GHB; 100 mg/kg, i.p., daily for 4 weeks) on the recovery of contractions of the distal hindlimb extensors and flexors after compression of the sciatic nerve in the rat. We also analyzed the dynamics of the functional sciatic index (FSI) and of the recovery of the contractile force (the latter index was estimated using tensometric transducers). In the control group, rats were also subjected to traumatic compression of the sciatic nerve but with no GHB injections. In the experimental GHB group, the recovery of the muscle activity was accelerated on the side of compression of the nerve; the rates of such recovery in various muscles were dissimilar. The indices of contractile activity of extensors of the foot and toes recovered on the 4th week (8 weeks earlier than in the control), while the contractile force of the toe flexors recovered on the 3rd week (7 weeks earlier). Within the late time intervals (10th and 12th weeks) after surgical intervention, the contractile force of the right foot extensors increased (by 40 and 30%, respectively, as compared with the control), while the flexors demonstrated only 5 and 4% increases. In the left (intact) hindlimb, the contractile force of flexors and extensors, beginning from the 3rd week, increased significantly (by 20-40%, as compared with the control). Within the late time intervals after traumatization, the GHB-influenced muscle contraction was not only greater than that in the control but also exceeded the initial values before surgical intervention. Therefore, the effect of GHB with respect to the contralateral (intact) hindlimb was manifested as an increment of the contractile force (in extensors, by 26 and 20% on the 10th and 12th week, and in flexors, respectively, by 48 and 36%). The probable mechanisms underlying the positive effect of GHB on the processes of recovery of the functional activity of peripheral nerves and muscle contractile force are discussed.

Comparative study of peripheral neuropathy and nerve regeneration in NOD and ICR diabetic mice

The non-obese diabetic (NOD) mouse was suggested as an adequate model for diabetic autonomic neuropathy. We evaluated sensory-motor neuropathy and nerve regeneration following sciatic nerve crush in NOD males rendered diabetic by multiple low doses of streptozotocin, in comparison with similarly treated Institute for Cancer Research (ICR) mice, a widely used model for type I diabetes. Neurophysiological values for both strains showed a decline in motor and sensory nerve conduction velocity at 7 and 8 weeks after induction of diabetes in the intact hindlimb. However, amplitudes of compound muscle and sensory action potentials (CMAPs and CNAPs) were significantly reduced in NOD but not in ICR diabetic mice. Morphometrical analysis showed myelinated fiber loss in highly hyperglycemic NOD mice, but no significant changes in fiber size. There was a reduction of intraepidermal nerve fibers, more pronounced in NOD than in ICR diabetic mice. Interestingly, aldose reductase and poly(ADP-ribose) polymerase (PARP) activities were increased already at 1 week of hyperglycemia, persisting until the end of the experiment in both strains. Muscle and nerve reinnervation was delayed in diabetic mice following sciatic nerve crush, being more marked in NOD mice. Thus, diabetes of mid-duration induces more severe peripheral neuropathy and slower nerve regeneration in NOD than in ICR mice.

Functional Reinnervation of the Distal Hindlimb Muscles in Rats after Compression of the Sciatic Nerve

Chronic experiments (12 weeks long) were carried out on two groups of pubertate-age Wistar rats. The sciatic nerve in one hindlimb was compressed, and complex analysis of the dynamics of functional reinnervation of the distal hindlimb muscles was performed. We used videorecording of images of the feet in the course of locomotion for estimation of the level of functional reinnervation of the toe extensors (the technique was modified in our laboratory) and a tensometric technique (measuring of the contraction force of the toe flexors and ankle extensors in the course of realization of the burrowing instinct by the animals). In the first group of animals (n = 21), we studied the dynamics of recovery of the force developed by the toe flexors and ankle extensors and of the functional sciatic index after sciatic nerve traumatization with no additional pharmacological influences. In the second group (n = 29), the same indices were analyzed, but after single injections of gamma-hydroxybutyrate (GHB) into animals before compression of the sciatic nerve and within early and late (3–4 and 14 weeks, respectively) terms after such intervention. Within the first week after compression, the smallest loss of activity was observed in the toe flexors, as compared with that in the toe and ankle extensors. Significant functional reinnervation of the ankle and toe extensors (to 52 and 87%, respectively) was observed on the 3rd to 7th weeks after the nerve injury. Functional reinnervation of the toe flexors was characterized by a relatively greater initial force of concentrations and more rapid recovery than those of the ankle and toe extensors. Practically complete recovery of the functions of the toe flexors was observed on the 10th week, while that of the ankle extensors was found on the 12th week. To make clearer possible reasons for differences between the processes of recovery of functions of the flexor and extensor muscles, we analyzed the effects of GHB on the contractile activity of these muscle groups within different time intervals after traumatization. In the second animal group, injection of GHB before operation resulted in drops of the muscle contraction force. After injury of the right sciatic nerve and injection of GHB on the 3rd posttraumatic week, recovery of the contraction force of all the examined muscles on the side of operation was more considerable. The functional loss of the muscle force in the left (intact) hindlimb in this case was 10 and 7% for the ankle extensors and toe flexors, respectively. Single injections of GHB within a late posttraumatic period resulted in a relative decrease in the contraction force of both flexors and extensors, and the functional loss was the greatest (35%) in the toe flexors.

An In Vitro Spinal Cord–Hindlimb Preparation for Studying Behaviorally Relevant Rat Locomotor Function

Although the spinal cord contains the pattern-generating circuitry for producing locomotion, sensory feedback reinforces and refines the spatiotemporal features of motor output to match environmental demands. In vitro preparations, such as the isolated rodent spinal cord, offer many advantages for investigating locomotor circuitry, but they lack the natural afferent feedback provided by ongoing locomotor movements. We developed a novel preparation consisting of an isolated in vitro neonatal rat spinal cord oriented dorsal-up with intact hindlimbs free to step on a custom-built treadmill. This preparation combines the neural accessibility of in vitro preparations with the modulatory influence of sensory feedback from physiological hindlimb movement. Locomotion induced by N-methyl D-aspartate and serotonin showed kinematics similar to that of normal adult rat locomotion. Changing orientation and ground interaction (dorsal-up locomotion vs ventral-up air-stepping) resulted in significant kinematic and electromyographic changes that were comparable to those reported under similar mechanical conditions in vivo. We then used two mechanosensory perturbations to demonstrate the influence of sensory feedback on in vitro motor output patterns. First, swing assistive forces induced more regular, robust muscle activation patterns. Second, altering treadmill speed induced corresponding changes in stride frequency, confirming that changes in sensory feedback can alter stride timing in vitro. In summary, intact hindlimbs in vitro can generate behaviorally appropriate locomotor kinematics and responses to sensory perturbations. Future studies combining the neural and chemical accessibility of the in vitro spinal cord with the influence of behaviorally appropriate hindlimb movements will provide further insight into the operation of spinal motor pattern-generating circuits.

Neuropeptide Y and age‐related development of hypertension in the rat

Essential hypertension is associated with increased sympathetic nerve activity in both animals and humans. We tested the hypothesis that neuropeptide Y (NPY) Y1 receptor activation was involved in the development of hypertension. In vivo infusions of NPY (15μg/kg) and Phenylephrine (PE, 10nmol/kg) to the intact hindlimb of spontaneously hypertensive rats (SHR) and their genetic control Wistar‐Kyoto (WKY) rats were performed in animals of 3, 5 and 7 weeks of age (n = 2–3 animals per strain per age). Femoral blood flow, blood pressure and heart rate were measured, while hindlimb vascular conductance (CHL), were calculated as an indicator of vascular responsiveness. Following phenylephrine (PE) infusions, CHL in the 7 week old SHR and WKY animals fell by 58.5% and 49.2% respectively (p&lt;0.05). The response remained depressed in both strains for at least 100s (p&lt;0.05). Compared with baseline, NPY infusion produced a 69% fall in CHL in the SHR that remained depressed for 100s (p&lt;0.05). No effect of NPY was observed in the 7 week WKY and there was minimal response to NPY in either the SHR and WKY animals at 5 or 3 weeks. Neither PE nor NPY affected CHL in the 3‐week old animals of either strain. Thus, while PE elicited similar responses in the SHR and the WKY animals, the sensitivity to exogenous NPY became pronounced with age in the SHR. These data provide the first evidence that the development of hypertension in the SHR involves an upregulation of the NPY system.

Biphasic change and disuse-mediated regression of canal network structure in cortical bone of growing rats

The canal network in cortical bone is an indispensable basis of bone vascularity, and its structure changes according to bone growth. Using monochromatic synchrotron radiation μCT (SRμCT), we evaluated the structural change of the canal network in growing rat tibiae and the response of this network to disuse. Tibiae were harvested from both hindlimbs of 9- and 14-week-old male Wistar rats subjected to unilateral sciatic neurectomy (SN) at 6 weeks of age (W9, n = 8; W14, n = 8) and from intact hindlimbs of 6-week-old rats (W6, n = 8). Images of distal diaphyseal segments were reconstructed by SRμCT with a voxel size of 5.83 μm and then translated into local mineral densities using a calibrated relation between linear absorption coefficients and the concentration of K 2HPO 4 solution. The canal network was segmented by simple thresholding at a bone mineral density of 0.82 g·cm − 3 and its structural properties were determined. In intact hindlimbs, the canal network showed a biphasic change with growth, as represented by increases followed by decreases in canal volume fraction (Ca.vol.f), the density of canals running longitudinally (Ca.num.d), and the density of canal connections (Ca.con.d): Ca.vol.f = 2.2, 3.1, and 1.8%, Ca.num.d = 77, 98, and 70 mm − 2 , and Ca.con.d = 18, 41, and 21 mm − 3 in W6, W9, and W14, respectively. In SN hindlimbs, bone growth deceleration was accompanied by a 16% smaller Ca.vol.f and a 22% smaller Ca.con.d in W9 and a 27% smaller Ca.vol.f, a 12% smaller Ca.num.d, and a 39% smaller Ca.con.d in W14 than those in intact hindlimbs. Furthermore, the canal branching structure became more treelike in SN hindlimbs. The effect of SN on the canal network appeared mainly in the periosteal sector of the anteriolateral cortex in W9 and spread throughout the cortex in W14. These findings will lead to a better understanding of microcirculation in cortical bone growth.

Neuroregeneration in Composite Tissue Allografts: Effect of Low-Dose FK506 and Mycophenolate Mofetil Immunotherapy

The immunosuppressant FK506 has been reported to increase the rate of peripheral nerve regeneration in nerve crush injury and nerve allograft models. The purpose of this study was to determine whether low doses of FK506 and mycophenolate mofetil had a neuroregenerative effect in revascularized peripheral nerve allografts in a rat hind limb transplantation model. Wistar Furth rat recipients received limbs from syngeneic Wistar Furth donors (group 1, n = 4) or from allogeneic August X Copenhagen Irish rat donors (group 2, n = 6). Wistar Furth recipients received limbs from August X Copenhagen Irish donors and were treated with FK506/mycophenolate mofetil for 5 months (group 3, n = 7). At the end of the follow-up period, histomorphometric analysis of sciatic and tibial nerves from transplanted and intact hind limbs was conducted. Sciatic and tibial nerves were examined at the level of coaptation and near the neuromuscular junction, respectively. Transplanted limbs in groups 1 and 3 completed the study without rejection, while the limbs in group 2 were rejected within a few days. Sciatic and tibial nerve analysis in groups 1 and 3 limbs showed myelinated axons of various diameters but in significantly fewer numbers than in nontransplanted contralateral nerves. The number and size of myelinated axons of transplanted nerves at corresponding levels were not significantly different between syngeneic and allogeneic (FK506/mycophenolate mofetil-treated) transplants. The authors conclude that long-term neuroregeneration of revascularized peripheral nerves using low-dose FK506/mycophenolate mofetil was similar to that of syngeneic transplants. The occurrence of acute rejection episodes with low-dose FK506/mycophenolate mofetil did not appear to benefit nor impair neuroregeneration.

Monochromatic synchrotron radiation μCT reveals disuse-mediated canal network rarefaction in cortical bone of growing rat tibiae

The purpose of this study was to demonstrate the ability of computed microtomography based on monochromatic synchrotron radiation (SRmuCT) in microstructural analysis of cortical bone. Tibial diaphyses of growing rats (14 wk, n = 8) undergoing unilateral sciatic neurectomy 8 wk before study were imaged with spatial volume resolution of 5.83 x 5.83 x 5.83 microm3 by SRmuCT (20 keV) at the synchrotron radiation facility (SPring-8). Reconstructed image data were translated into local mineral densities by using a calibrated linear relationship between linear absorption coefficients and concentrations of homogeneous K2HPO4 solution. Pure bone three-dimensional images, produced by simple thresholding at a bone mineral density of 0.82 g/cm3, were analyzed for macro- and microscopic structural properties. In neurectomized hindlimbs, cortical canal network rarefaction as well as bone atrophy were found. The former was characterized by 30% smaller porosity, 11% smaller canal density in transverse section, and 38% smaller canal connectivity density than those in contralateral bone. On the other hand, no difference was found in bone mineral density between neurectomized and intact hindlimbs (1.37 vs. 1.36 g/cm3). In conclusion, SRmuCT is a promising method for the three-dimensional analysis of cortical microstructure and the degree of mineralization in small animals.

Femoral dimensions and mid‐thigh circumference in Alligator mississippiensis

The mid-thigh circumference of the intact hindlimb in Alligator mississippiensis is tightly correlated with transverse dimensions of the proximal and distal articular surfaces of the femur, and with minimum midshaft femoral circumference. Maximum diameter of the proximal articular end, width across the distal articular end, and midshaft circumference are the best femoral predictors of circumference of the intact thigh. Regression equations of mid-thigh circumference against these femoral dimensions can be used to estimate the transverse dimensions of the intact hindlimb in extinct crocodylian-like archosaurs.

Connections of neurons in the lumbar ventral horn of spinal cord are altered after long-standing limb loss in a macaque monkey

Explanations for the massive reorganization in primary motor cortex, M1, after limb amputation typically focus on processes that occur in cortex. Few have investigated whether changes in more peripheral parts of the pathway might also play a role in the reorganization. In the present study, we examined the integrity and connectivity of the spinal cord motoneurons in a macaque monkey (Macaca mulatta) that lost a hindlimb as a result of accidental injury more than 3.5 years earlier. To label motoneurons, multiple small injections of a neuroanatomical tracer were placed in the muscles of the hip just adjacent to the stump of the amputated leg, and in matched locations in the opposite side for control purposes. Injections of a second tracer were made in the intact foot. In the ventral horn that related to the intact hindlimb, motoneurons labeled by the hip injections were concentrated rostral and ventromedial to those labeled by the foot injections. Hip injections on the side of the amputation labeled neurons that were located well beyond the normal territory for motoneurons related to the hip and into the zone normally occupied by neurons projecting to the foot. Labeled motoneurons innervating the intact limb were significantly larger than neurons on the side of the amputation (x = 2410 and 2061 µm2, respectively). The findings suggest that many neurons survived the long-standing amputation, and made new connections with remaining intact muscles. These new patterns of connectivity likely contribute to the reorganization of motor cortex in amputees, and perhaps to abnormal behaviors like those reported by human amputees.

A substance P receptor (NK 1) antagonist enhances the widespread osteoporotic effects of sciatic nerve section

The long-term effects of sciatic nerve section on bone mineral density (BMD) were studied using dual-energy X-ray absorptiometry (DEXA) in skeletally mature rats. Unilateral sciatic neurectomy caused the rapid loss of cancellous bone in the proximal and distal femur and tibia in the ipsilateral hindlimb and, to a lesser extent, in the contralateral intact hindlimb. The reduction in BMD rapidly progressed for 4 weeks after sciatic section and then gradually stabilized with no evidence of recovery at 12 weeks. The development of osteoporosis in the contralateral intact hindlimb was a novel finding. There was no evidence of disuse in the normal contralateral hindlimb after unilateral sciatic section; grid-crossing activity over a 24-h interval was unchanged and there was no reduction in weight bearing on the contralateral normal hindpaw during the stance phase of ambulation. Unilateral peripheral nerve lesions have well-documented effects on substance P content and function in the corresponding contralateral intact nerve. We hypothesized that after sciatic section a reduction in substance P signaling might contribute to bone loss in the contralateral hindlimb. Daily administration of the substance P receptor (NK 1) antagonist LY303870 for 2 weeks caused significant loss of cancellous bone in the denervated and the contralateral hindlimb, evidence that substance P signaling sustained bone density after nerve section. After sciatic neurectomy there was a 33% reduction in sciatic nerve stimulation-evoked extravasation in the contralateral intact hindlimb, indicating transmedian inhibition of substance P signaling after nerve injury. Furthermore, there was a 50% reduction in the substance P content in both tibias after unilateral sciatic section. Collectively, these data support the hypothesis that a widespread reduction in substance P content in bone contributes to the osteoporotic effects of sciatic neurectomy and that residual substance P signaling maintains bone integrity after nerve section in both the denervated and contralateral intact hindlimb.

Lipoprotein lipase activity in skeletal muscles of the rat: effects of denervation and tenotomy.

The effects of denervation, tenotomy, or tenotomy with simultaneous denervation on the activity of heparin-releasable and intracellular, residual lipoprotein lipase (LPL) and triacylglycerol (TG) content were examined in rat skeletal muscles. An influence of muscle electrostimulation on denervated and tenotomized muscles was also evaluated. Activity of both LPL fractions was decreased in denervated and/or tenotomized soleus and red portion of gastrocnemius muscles. It was accompanied by a slight elevation of the intracellular TG content. Electrostimulation increased activities of both fractions of LPL in red muscles from intact hindlimbs. In stimulated denervated muscles without or with simultaneous tenotomy, activity of two LPL fractions was also enhanced, but control values were reached only in denervated soleus muscle. Electrical stimulation had no pronounced effect on LPL activity in tenotomized muscles. In conclusion, denervation and/or tenotomy decreases LPL activity in red muscles, indicating reduction of the muscle potential to utilize circulating TG. Electrostimulation only partly restores the diminished LPL activity in denervated muscles, without any effect in tenotomized ones. Thus, to maintain LPL activity in resting muscle, intact innervation and tension are needed.

Accurate assessment of in situ isometric contractile properties of hindlimb plantar and dorsal flexor muscle complex of intact mice.

An isometric torque sensor for measuring in situ contractions of plantar or dorsal flexors of intact mouse hindlimb has been developed and evaluated. With this device, muscle torque can be accurately measured within the range of -14 mN.m to +14 mN.m. Special attention was paid to fixation of the mouse hindlimb to the measurement device. Halothane-anaesthetized Swiss wild-type mice were positioned on the thermostatic measurement platform, and fixated with a hip and foot fixation system. The novel fixation unit was evaluated by measuring knee and ankle displacements during a contraction. A mathematical muscle model was used to quantify the effects of these displacements on the contractile parameters. Measured ankle and knee displacement, due to non-absolute fixation. resulted in a calculated muscle fibre shortening of 2.5%. Simulations of a contraction with this degree of fibre shortening, using the mathematical muscle model, showed only minor effects on maximal torque generation and the temporal parameters (half-relaxation time and 10-50% rise time). Furthermore, we showed that muscle torque in our set-up is hardly affected by eccentricity between ankle and measurement axis. Measured tetanic muscle torques of intact dorsal and plantar flexors were 3.2+/-0.4 mN.m and 11.8+/-1.6 mN.m, respectively. The half-relaxation time of plantar flexors was significantly higher than that of dorsal flexors (12.9+/-2.7 ms versus 8.8+/-1.2 ms), whereas the 10-50% rise time was longer in plantar (14.9+/-0.6 ms) than in dorsal (11.8+/-2.0 ms) flexors.

Effect of tibial bone resection on the development of fast- and slow-twitch skeletal muscles in foetal sheep.

To determine if longitudinal bone growth affects the differentiation of fast- and slow-twitch muscles, the tibial bone was sectioned at 90 days gestation in foetal sheep so that the lower leg was permanently without structural support. At 140 days (term is approximately 147 days) the contractile properties of whole muscles, activation profiles of single fibres and ultrastructure of fast- and slow-twitch muscles from the hindlimbs were studied. The contractile and activation profiles of the slow-twitch soleus muscles were significantly affected by tibial bone resection (TIBX). The soleus muscles from the TIBX hindlimbs showed: (1) a decrease in the time to peak of the twitch responses from 106.2 +/- 10.7 ms (control, n = 4) to 65.1 +/- 2.48 ms (TIBX, n = 5); (2) fatigue profiles more characteristic of those observed in the fast-twitch muscles: and (3) Ca2+ - and Sr2+ -activation profiles of skinned fibres similar to those from intact hindlimbs at earlier stages of gestation. In the FDL, TIBX did not significantly change whole muscle twitch contraction time, the fatigue profile or the Ca2+ - and Sr2+ -activation profiles of skinned fibres. Electron microscopy showed an increased deposition of glycogen in both soleus and FDL muscles. This study shows that the development of the slow-twitch phenotype is impeded in the absence of the physical support normally provided by the tibial bone. We suggest that longitudinal stretch is an important factor in allowing full expression of the slow-twitch phenotype.