The rabbit brachial plexus as a model for nerve injury and repair Part 1: Anatomic study of the biceps and triceps innervation.

PURPOSE: Animal models can be helpful in evaluating new surgical strategies for brachial plexus reconstruction. While several groups have already used the rabbit brachial plexus to model injury, reports conflict in anatomic detail and do not identify a nerve-muscle pair to measure motor function recovery after reconstruction. The purpose of the current study is to describe the innervations of the biceps and triceps muscles in rabbits, which are both amenable to study in brachial plexus injury models. MATERIALS AND METHODS: Thirteen rabbits weighing 2-2.5 kg were anesthetized. Six rabbits were sacrificed and dissected using loupe and microscope magnification to understand the overall morphology of the brachial plexus. Seven rabbits underwent electrophysiologic investigation. A bipolar nerve stimulator was used to systematically stimulate the roots, trunks and divisions, and nerve branches of the rabbit brachial plexus and compound muscle action potential was used to record muscle response. Nerve length and width measurements were not recorded. RESULTS: Roots contributing to the brachial plexus were C5, C6, C7, C8, and T1. In contrast to other anatomical studies, T2 did not contribute to the brachial plexus. The triceps was innervated by the radial nerve, which received contributions from C6 (1.6 mA), C7 (1.9 mA), C8, and T1 (12.2 mA).The biceps had dual innervation (proximally and distally). The proximal branch received contributions from C6 (3.5 mA) and C7 (5mA). The distal portion was innervated by a branch from the median nerve, which received innervation from C6, C7, C8, and T1. CONCLUSIONS: The overall structure of rabbit brachial plexus is described and innervation of the biceps and triceps is described in detail. This anatomic investigation will form the basis of a future brachial plexus model of injury and repair.

Outcomes of Vascularized Bone Allotransplantation with Surgically Induced Autogenous Angiogenesis in a Large Animal Model: Bone Healing, Remodeling, and Material Properties.

BACKGROUND: Bone vascularized composite allotransplantation (VCA) is a possible alternative for the treatment of large bone defects. Clinical application of VCAs is limited by the need for life-long immunosuppression (IS). We report an alternative method to maintain bone allotransplant viability in a large animal model without the need for life-long IS by using autogenous vessel implantation. METHODS: Fourteen bone only VCAs were transplanted in a porcine tibia defect model with short-term IS. Two groups were used to evaluate the effect of the implantation of an autogenous arteriovenous (AV)-bundle, therefore the only difference between the groups was the patency of the AV-bundle. We radiographically evaluated bone healing and allogenic pedicle patency. AV-bundle patency and union were evaluated with micro-CT. Bone remodeling was assessed with histomorphometry and material properties were evaluated with axial compression testing and cyclic reference point indentation. RESULTS: Two subjects did not reach the final time point. Twelve tibiae healed proximally, and nine at the distal transplant-bone interface. Bone allotransplants showed their viability in the first 4 to 6 weeks by significant periosteal bridging arising from the transplant and maintained pedicle patency. Bone material properties were not affected by the implantation of an AV-bundle when compared with ligated AV-bundle controls, but diminished compared with normal bone. Significantly higher bone formation rates resulted from the implantation of a patent AV-bundle. CONCLUSION: New periosteal bone formation and subsequent bone healing result from blood flow through the microsurgically repaired nutrient blood supply, demonstrated by maintained allogenic pedicle patency. The implantation of a patent autogenous AV-bundle has no adverse effect on material properties, but a positive effect on bone remodeling of endosteal surfaces despite thrombosis of the allogenic pedicle. Bone material properties change after transplantation compared with normal bone, although 20-weeks survival time is relatively short for the final evaluation of bone material properties.

Brachial plexus nerve injury and repair in a rabbit model part II: Does middle trunk injury result in loss of biceps function while repair results in recovery of biceps function.

INTRODUCTION: There is conflicting anatomic and innervation data regarding the rabbit brachial plexus injury model. This study aims to validate a rabbit brachial plexus injury model. We hypothesize the middle trunk (C6, C7) is the primary innervation of the biceps, and when cut and unrepaired, would demonstrate lack of recovery and when repaired would demonstrate evidence of recovery. MATERIALS AND METHODS: Twenty two male New Zealand white rabbits (3-4 kg) underwent unilateral surgical division of the middle trunk. Five rabbits were randomly assigned to the "no-repair" group while the remaining 17 rabbits underwent direct coaptation ("repair" group). Rabbits were followed for 12 weeks, with ultrasound measurement of biceps cross-sectional area performed preoperatively, and at 4, 8, and 12 weeks postoperatively. At a euthanasia procedure, bilateral compound muscle action potential (CMAP) and isometric tetanic force (ITF) were measured. Bilateral biceps muscles were harvested and wet muscle weight was recorded. The operative side was expressed as a percentage of the non-operated side, and differences between the no repair and repair rabbits were statistically compared. RESULTS: The repair group demonstrated significantly higher CMA (23.3 vs. 0%, p < .05), ITF (25.6 vs. 0%, p < .05), and wet muscle weight (65.8 vs. 52.0%, p < .05) as compared to the unrepaired group. At 4 weeks postoperatively, ultrasound-measured cross-sectional area of the biceps demonstrated atrophy in both groups. At 12 weeks, the repair group had a significantly larger cross-sectional area as compared to the no-repair group (89.1 vs. 59.3%, p < .05). CONCLUSIONS: This injury model demonstrated recovery with repair and lack of function without repair. Longer survival time is recommended for future investigations.

Bone vascularized composite allotransplantation model in swine tibial defect: Evaluation of surgical angiogenesis and transplant viability.

INTRODUCTION: In prior small animal studies, we maintained vascularized bone allotransplant viability without long-term immunotherapy. Instead, an autogenous neoangiogenic circulation is created from implanted vessels, sufficient to maintain bone viability with only 2 weeks immunosupression. Blood flow is maintained despite rejection of the allogeneic vascular pedicle thereafter. We have previously described a large animal (swine) pre-clinical model, reconstructing tibial defects with vascularized tibial allotransplants. In this manuscript, autologous angiogenesis is evaluated in this model and correlated with bone viability. MATERIALS AND METHODS: Allogeneic tibial segments were transplanted across a major swine leukocyte antigen mismatch. Microvascular repair of the bone VCA pedicle was combined with intraosseous implantation of an autogenous arteriovenous (AV) bundle. The bundle was ligated in group 1 (n = 4), and allowed to perfuse in group 2 (n = 4). Three-drug immunotherapy was given for 2 weeks. At 16 weeks micro-CT angiography quantified neoangiogenic vessel volume. Bone viability, rejection grade, and bone healing were analyzed. RESULTS: A substantial neoangiogenic circulation developed from the implanted AV-bundle in group 2, with vessel density superior to ligated AV-bundle controls (0.11 ± 0.05 vs. 0.01 ± 0.01, P = .029). Bone allotransplant viability was also significantly enhanced by neoangiogenesis (78.7 ± 4.4% vs. 27.7 ± 5.8%, P = .028) with higher bone healing scores (21.4 ± 2.9 vs. 12.5 ± 3.7, P = .029). Ligated control tibias demonstrated disorganized bone morphology and higher local inflammation (P = .143). CONCLUSION: Implantation of autogenous AV bundles into vascularized bone allotransplants resulted in the rapid formation of a neoangiogenic autogenous blood supply in a swine tibia model that maintained bone viability, improved bone healing, and minimized rejection.

Validation of Isometric Tetanic Force as a Measure of Muscle Recovery After Nerve Injury in the Rabbit Biceps.

PURPOSE: The purpose of this study was to describe and validate a technique for measurement of isometric tetanic force (ITF) in the rabbit biceps muscle. MATERIALS AND METHODS: Eighteen New Zealand White rabbits were randomized to test either the right side or the left side first. Under propofol anesthesia, the brachial plexus and biceps brachii were exposed. The middle trunk (C6, C7) was secured in a bipolar electrode. Compound muscle action potential (CMAP) was measured. The proximal, tendinous portion of the biceps was severed at the shoulder and clamped in a custom-made force transducer. Muscle preload and electrical stimulation variables were optimized to obtain the highest tetanic muscle contraction. Wet muscle weight (WMW) and nerve histomorphometry were analyzed. Statistical analysis was performed to determine side-to-side equivalence. RESULTS: The rabbit biceps muscle force demonstrated side-to-side equivalence with overlapping 95% confidence intervals (95% CI). The right side, expressed as a percentage of the left, averaged 99.69% (95% CI, 88.89%-110.5%). The WMW of the right expressed as a percentage of the left was 98.9% (95% CI, 95.8%-102%). CONCLUSIONS: The ITF is equivalent from side to side in the rabbit as demonstrated by the high degree of overlap in the 95% CIs for each side. The width of the 95% CI implies that there is more variability in the rabbit upper extremity than for the lower extremity of the rabbit or rat models, and researchers should take this into account when performing sample size estimates in pre-experimental planning. CLINICAL RELEVANCE: The rabbit biceps muscle ITF measurements can be used to measure motor recovery in a rabbit model of brachial plexus injury and compared with the contralateral uninjured side.

The effect of full dose composite tissue allotransplantation immunosuppression on allograft motor nerve regeneration in a rat sciatic nerve model.

BACKGROUND: The purpose of this study was to identify which triple immunosuppressive protocols, currently used for vascularized composite allotransplantation in the clinic, will have the best effect on motor function recovery following nerve allograft reconstruction. METHODS: Eighty-eight Lewis rats underwent a 1-cm sciatic nerve allograft transplantation and skin graft from 44 Brown-Norway rats. Group I received 0.9% isotonic saline (control); Group II, 2 mg/kg FK506; Group III, 1 mg/kg FK506 with 15 mg/kg mycophenolate mofetil (MMF); and Group IV, 2 mg/kg FK506 with 30 mg/kg MMF and prednisone. Each group consisted of 11 rats. After 12 weeks, motor function recovery was evaluated with isometric tetanic force, muscle mass, ankle contracture angle, electrophysiology, and nerve histomorphometry. Adequacy of immunosuppression was monitored with the transplanted skin graft. All data are expressed as a percentage of the contralateral side. RESULTS: Isometric tetanic force showed significantly better functional recovery in all groups treated with immunosuppression compared to control. Within the immunosuppression groups no significant difference was found: 42.1 ± 6.4% (Group I), 56.1 ± 12.4% (Group II), 58.4 ± 10.7% (Group III), and 61.3 ± 11.2% (Group IV). Group IV was superior to all other groups regarding ankle contracture (P < .05) and electrophysiology (P < .001). Skin graft rejection occurred in 41 and 0% (Groups III and IV, respectively). CONCLUSIONS: FK506 significantly enhanced motor recovery after allograft reconstruction. This effect was comparable between combination treatment (low-dose FK506 and MMF) and triple therapy (high-dose FK506 and MMF plus prednisolone). However, triple therapy was more effective in suppressing skin rejection.

A new porcine vascularized tibial bone allotransplantation model. Anatomy and surgical technique.

INTRODUCTION: Vascularized bone allotransplantation may provide new options for reconstruction of segmental defects if problems of long-term immune modulation can be solved. The current literature lacks an orthotopic large animal model, limited to bone and without the confounding effects of other tissue types, permitting a multifaceted evaluation before new methods are used clinically. The purpose of this study was to develop a large animal model for vascularized bone allotransplantation. MATERIALS AND METHODS: Eight porcine hind limbs were dissected. Length, diameter, and location of all hindlimb vessels were measured and a single nutrient vessel supplying the tibial diaphysis identified enabling its use as a vascularized bone allotransplant. Four Yucatan minipigs were divided into two pairs with a major swine leukocyte antigen mismatch. A 3.5 cm tibial segment including its nutrient pedicle was raised simultaneously from each pig and transplanted into the matched defect of the other animal. Microarterial anastomosis of the pedicle and 3-drug immunosuppression maintained VCA viability. Bone healing and limb function were followed for 16 weeks. RESULTS: A consistent tibia diaphyseal nutrient artery arose from the caudal tibial artery to enter bone a mean 2.8 mm distal to the tibial tubercle with a pedicle length of 6.6 ± 3.3 mm and diameter of 1.6 ± 0.2 mm. Using this pedicle, we reconstructed a 3.5 cm tibial defect with a vascularized bone allotransplant in four animals. Immediate weightbearing as well as progressive bone healing was demonstrated. CONCLUSION: We have developed a vascularized tibial bone allotranplantation large-animal model suitable for future bone-only allotranplantation research in mini-pigs.

Comparable functional motor outcomes after repair of peripheral nerve injury with an elastase-processed allograft in a rat sciatic nerve model.

BACKGROUND: A bridging nerve autograft is the gold standard for the repair of segmental nerve injury that cannot be repaired directly. However, limited availability and donor site morbidity remain major disadvantages of autografts. Here, a nerve allograft decellularized with elastase was compared with an autograft regarding functional motor outcome in a rat sciatic segmental nerve defect model. Furthermore, the effect of storage on this allograft was studied. METHODS: Sixty-six Lewis rats (250-300 g) underwent a 10-mm sciatic nerve reconstruction using either a cold- (n = 22) or frozen-stored (n = 22) decellularized nerve allograft or an autograft (n = 22). Sprague-Dawley rats (300-350 g) served as full major histocompatibility complex-mismatched donors. Functional motor outcome was evaluated after 12 and 16 weeks. Ankle angle, compound muscle action potential (CMAP), isometric tetanic force, wet muscle weight, and histomorphometry were tested bilaterally. RESULTS: For CMAP and isometric tetanic force, no significant differences were observed between groups. In contrast, for ankle angle, histomorphometry and muscle weight, the cold-stored allograft performed comparable to the autograft, while the frozen-stored allograft performed significantly inferior to the autograft. At week 16, ankle angle was 88.0 ± 3.1% in the cold-stored group, 77.4 ± 3.6% in the frozen-stored group, and 74.1 ± 3.1% in the autograft group (P < .001); At week 16, the muscle weight showed a recovery up to 71.1 ± 4.8% in the autograft group, 67.0 ± 6.6% in the cold-stored group, and 64.7 ± 3.7% in the frozen-stored group (P < .05). CONCLUSIONS: A nerve allograft decellularized with elastase, if stored under the right conditions, results in comparable functional motor outcomes as the gold standard, the autograft.

Optimizing decellularization techniques to create a new nerve allograft: an in vitro study using rodent nerve segments.

OBJECTIVE Commercially available processed nerve allografts have been shown to be inferior to autografts in previous animal studies. The authors hypothesized that combining different processing and storage techniques will result in improved nerve ultrastructure preservation, lower immunogenicity, and minimized cellular debris. Different processing protocols were evaluated using chemical detergents, enzymes, and irradiation, with the addition the of enzyme elastase, were used. Additionally, the difference between cold and frozen storage was investigated. The goal of this study was to create an optimized nerve allograft. METHODS Fifty rat nerves were decellularized with modifications of previous protocols and the addition of elastase. Subsequently, the nerve segments were stored at either 4°C or -80°C. Both processed and fresh control nerves were analyzed with confocal microscopy using immunohistochemical staining on the basal lamina (laminin γ-1), Schwann cells (S100 protein), and immunogenicity using major histocompatibility complex-I (MHCI) staining. Morphology of the ultrastructure and amount of cellular debris were analyzed on cross-sections of the nerves stained with toluidine blue and H & E, and by using electron microscopy. RESULTS Nerve ultrastructure was preserved with all decellularization protocols. Storage at -80°C severely altered nerve ultrastructure after any decellularization method. Elastase was found to significantly reduce the immunogenicity and amount of Schwann cells, while maintaining good structural properties. CONCLUSIONS Reduced immunogenicity, diminished cellular debris, and the elimination of Schwann cells was observed when elastase was added to the nerve processing while maintaining ultrastructure. Storage at -80°C after the decellularization process heavily damaged the nerve ultrastructure as compared with cold storage. Further in vivo studies are needed to prove the nerve regenerative capacity of these optimized allografts.

Motor Nerve Recovery in a Rabbit Model: Description and Validation of a Noninvasive Ultrasound Technique.

PURPOSE: To develop and validate a noninvasive ultrasound technique for the longitudinal analysis of functional recovery after segmental peroneal nerve reconstruction in a rabbit model. METHODS: Twelve male New Zealand White rabbits underwent a 1-cm peroneal nerve autograft reconstruction. Ultrasound measurements were performed before surgery and at 1, 2, 4, 8, 12, and 16 weeks postoperatively. All rabbits were managed with manual restraint for the ultrasound procedure, avoiding the risks of anesthetics. At 12 and 16 weeks, we evaluated functional recovery using compound muscle action potential, isometric tetanic force measurements, wet muscle weight, and nerve histomorphometry. Data were compared with ultrasound measurements by calculating the Pearson correlation coefficient. We determined intra-rater and inter-rater reliability of the ultrasound measurements. RESULTS: Ultrasound demonstrated good correlation with isometric tetanic force measurements and wet muscle weight, good correlation with nerve histomorphometry, and moderate correlation with compound muscle action potential. Both intra-rater and inter-rater reliability of the ultrasound technique was excellent. CONCLUSIONS: Ultrasound analysis of the tibialis anterior muscle provided a reliable method for analysis of functional recovery in a rabbit peroneal nerve reconstruction model. The noninvasive nature allowed for longitudinal follow-up within the same animal and measurement of early recovery without the use of anesthesia. CLINICAL RELEVANCE: Application of this noninvasive technique can reduce the variability and sample size necessary in peripheral nerve reconstruction studies and may provide an ideal tool for comparative studies in larger animal models.

Effect of Vascular Endothelial Growth Factor Administration on Nerve Regeneration after Autologous Nerve Grafting.

BACKGROUND: The aim of this study was to evaluate the effect of vascular endothelial growth factor (VEGF) administration around the autologous nerve graft on nerve recovery in a rat model. METHODS: A total of 69 rats were randomly divided into three experimental groups. A 10-mm sciatic nerve defect was made and reconstructed with the reversed nerve segment. Group I received an osmotic pump with saline, group II received an osmotic pump with VEGF, and group III added a silicone tube around the nerve graft to decrease the surrounding blood supply. Nine animals in each group were sacrificed on day 3 to evaluate improvement in new vessel formation. In each group 14 animals were sacrificed at 16 weeks after the initial procedure to evaluate the functional motor nerve regeneration using compound muscle action potential, isometric tetanic force, wet muscle weight, and nerve histomorphometry. RESULTS: The average vascular density on day 3 was 10.7% in group I, 21.4% in group II, and 0.9% in group III. These differences were significant. However, the average maximum isometric tetanic force at 16 weeks was 54.4% in group I, 57.5% in group II, and 47.6% in group III. No difference was found with or without VEGF administration. Histomorphometric analysis was also not significantly different between the groups. CONCLUSIONS: New vessel formation on autologous nerve graft was enhanced by VEGF administration. However, the neovascularization effect of VEGF administration did not translate into better motor nerve recovery.

Vascularized bone transplant chimerism mediated by vascular endothelial growth factor.

BACKGROUND: Vascular endothelial growth factor (VEGF) induces angiogenesis and osteogenesis in bone allotransplants. We aim to determine whether bone remodeling in VEGF-treated bone allotransplants results from repopulation with circulation-derived autogenous cells or survival of allogenic transplant-derived cells. METHODS: Vascularized femoral bone transplants were transplanted from female Dark Agouti rats (DA;RT1(a) ) to male Piebald Viral Glaxo (PVG;RT1(c) ). Arteriovenous bundle implantation and short-term immunosuppression were used to maintain cellular viability. VEGF was encapsulated in biodegradable microspheres and delivered intramedullary in the experimental group (n = 22). In the control group (n = 22), no VEGF was delivered. Rats were sacrificed at 4 or 18 weeks. Laser capture microdissection of bone remodeling areas was performed at the inner and outer cortex. Sex-mismatched genes were quantified with reverse transcription-polymerase chain reaction to determine the amount of male cells to total cells, defined as the relative expression ratio (rER). RESULTS: At 4 weeks, rER was significantly higher at the inner cortex in VEGF-treated transplants as compared to untreated transplants (0.622 ± 0.225 vs. 0.362 ± 0.081, P = 0.043). At 4 weeks, the outer cortex in the control group had a significantly higher rER (P = 0.038), whereas in the VEGF group, the inner cortex had a higher rER (P = 0.015). Over time, in the outer cortex the rER significantly increased to 0.634 ± 0.106 at 18 weeks in VEGF-treated rats (P = 0.049). At 18 weeks, the rER was >0.5 at all cortical areas in both groups. CONCLUSIONS: These in vivo findings suggest a chemotactic effect of intramedullary applied VEGF on recipient-derived bone and could imply that more rapid angiogenesis of vascularized allotransplants can be established with microencapsulated VEGF.

Cell lineage in vascularized bone transplantation.

BACKGROUND: The biology behind vascularized bone allotransplantation remains largely unknown. We aim to study cell traffic between donor and recipient following bone auto-, and allografting. METHODS: Vascularized femoral transplantation was performed with arteriovenous bundle implantation and short-term immunosuppression. Twenty male Piebald Virol Glaxo (PVG; RT1(c) ) rats received isotransplants from female PVG (RT1(c) ) rats and 22 male PVG rats received allografts from female Dark Agouti rats (DA, RT1(a) ), representing a major histocompatibility mismatch. Both groups were randomly analyzed at 4 or 18 weeks. Bone remodeling areas (inner and outer cortical samples) were labeled and laser capture microdissected. Analysis of sex-mismatch genes by real-time reverse transcription-polymerase chain reaction provided the relative Expression Ratio (rER) of donor (female) to recipient (male) cells. RESULTS: The rER was 0.456 ± 0.266 at 4 weeks and 0.749 ± 0.387 at 18 weeks (p = 0.09) in allotransplants. In isotransplants, the rER was 0.412 ± 0.239 and 0.467 ± 0.252 at 4 and 18 weeks, respectively (p = 0.21). At 4 weeks, the rER at the outer cortical area of isotransplants was significantly lower in isotransplants as compared with allotransplants (0.247 ± 0.181 vs. 0.549 ± 0.184, p = 0.007). Cells in the inner and outer cortical bone remodeling areas in isotransplants were mainly donor derived (rER < 0.5) at 18 weeks, whereas allotransplants contained mainly recipient-derived cells (rER > 0.5) at 18 weeks. CONCLUSIONS: Applying novel methodology, we describe detailed cell traffic in vascularized bone transplants, elaborating our comprehension on bone transplantation.

Does the addition of a nerve wrap to a motor nerve repair affect motor outcomes?

The purpose of this study was to evaluate the effect of wrapping bioabsorbable nerve conduit around primary suture repair on motor nerve regeneration in a rat model. Forty rats were randomly divided into two experimental groups according to the type of repair of the rat sciatic nerve: group I had primary suture repair; group II had primary suture repair and bioabsorbable collagen nerve conduit (NeuraGen® 1.5 mm, Integra LifeSciences Corp., Plainsboro, NJ) wrapped around the repair. At 12 weeks, no significant differences in the percentage of recovery between the two groups were observed with respect to compound muscle action potentials, isometric muscle force, and muscle weight (P = 0.816, P = 0.698, P = 0.861, respectively). Histomorphometric analysis as compared to the non-operative sites was also not significantly different between the two groups in terms of number of myelinated axons, myelinated fiber area, and nerve fiber density (P = 0.368, P = 0.968, P = 0.071, respectively). Perineural scar tissue formation was greater in primary suture repair group (0.36 ± 0.15) than in primary repair plus conduit wrapping group (0.17 ± 0.08). This difference was statistically significant (P < 0.001). Wrapping bioabsorbable nerve conduit around primary nerve repair can decrease perineural scar tissue formation. Although the scar-decreasing effect of bioabsorbable nerve wrap does not translate into better motor nerve recovery in this study, it might have an effect on the functional outcome in humans where scar formation is much more evident than in rats.

Fibroblast growth factor-2 and vascular endothelial growth factor mediated augmentation of angiogenesis and bone formation in vascularized bone allotransplants.

We previously demonstrated recipient-derived neoangiogenesis to maintain viability of living bone allogeneic transplants without long-term immunosuppression. The effect of cytokine delivery to enhance this process is studied. Vascularized femur transplantation was performed from Dark Agouti to Piebald Virol Glaxo rats. Poly(d,l-lactide-co-glycolide) microspheres loaded with buffer (N = 11), basic fibroblast growth factor (FGF2) (N = 10), vascular endothelial growth factor (VEGF) (N = 11), or both (N = 11) were inserted intramedullarly alongside a recipient-derived arteriovenous bundle. FK-506 was administered for 2 weeks. At 18 weeks, bone blood flow, microangiography, histologic, histomorphometric, and alkaline phosphatase measurements were performed. Bone blood flow was greater in the combined group than control and VEGF groups (P = 0.04). Capillary density was greater in the FGF2 group than in the VEGF and combined groups (P < 0.05). Bone viability, growth, and alkaline phosphatase activity did not vary significantly between groups. Neoangiogenesis in vascularized bone allotransplants is enhanced by angiogenic cytokine delivery, with results using FGF2 that are comparable to isotransplant from previous studies. Further studies are needed to achieve bone formation similar to isotransplants.

Description and validation of isometric tetanic muscle force test in rabbits.

Isometric tetanic muscle force has been described in a rat model to evaluate motor recovery in a segmental sciatic nerve defect reconstructions. However, to test longer nerve defects, an alternative and larger animal model is necessary. The purpose of this study is to describe and validate a technique for isometric force measurement of the tibialis anterior (TA) muscle in New Zealand rabbits. Muscle preload and electrical stimulation parameters were optimized to obtain the highest tetanic contraction bilaterally in 10 animals. Electrophysiology, muscle weight, peroneal nerve length, and histomorphometry were also analyzed. Only the peroneal nerve length and the ratio of highest muscle force/muscle weight demonstrated the equivalence between the sides. A small variability of TA muscle force and TA muscle weight was observed between the sides suggesting dominance. Optimization of electrical stimulation and preload as well as the use of correct anesthesia were fundamental to acquire the highest muscle force.

Knee joint transplantation combined with surgical angiogenesis in rabbits--a new experimental model.

PURPOSE: We have previously described a means to maintain bone allotransplant viability, without long-term immune modulation, replacing allogenic bone vasculature with autogenous vessels. A rabbit model for whole knee joint transplantation was developed and tested using the same methodology, initially as an autotransplant. MATERIALS/METHODS: Knee joints of eight New Zealand White rabbits were elevated on a popliteal vessel pedicle to evaluate limb viability in a nonsurvival study. Ten additional joints were elevated and replaced orthotopically in a fashion identical to allotransplantation, obviating only microsurgical repairs and immunosuppression. A superficial inferior epigastric facial (SIEF) flap and a saphenous arteriovenous (AV) bundle were introduced into the femur and tibia respectively, generating a neoangiogenic bone circulation. In allogenic transplantation, this step maintains viability after cessation of immunosuppression. Sixteen weeks later, X-rays, microangiography, histology, histomorphometry, and biomechanical analysis were performed. RESULTS: Limb viability was preserved in the initial eight animals. Both soft tissue and bone healing occurred in 10 orthotopic transplants. Surgical angiogenesis from the SIEF flap and AV bundle was always present. Bone and joint viability was maintained, with demonstrable new bone formation. Bone strength was less than the opposite side. Arthrosis and joint contractures were frequent. CONCLUSION: We have developed a rabbit knee joint model and evaluation methods suitable for subsequent studies of whole joint allotransplantation.

Surgical Angiogenesis of Decellularized Nerve Allografts Improves Early Functional Recovery in a Rat Sciatic Nerve Defect Model.

BACKGROUND: Surgical angiogenesis applied to nerve grafts has been suggested to enhance nerve regeneration after nerve injury. The authors hypothesized that surgical angiogenesis to decellularized nerve allografts would improve functional recovery in a rat sciatic nerve defect model. METHODS: Sixty Lewis rats were divided in three groups of 20 animals each. Unilateral sciatic nerve defects were repaired with (1) autografts, (2) decellularized allografts, and (3) decellularized allografts wrapped with a superficial inferior epigastric artery fascial flap to add surgical angiogenesis. Twelve and 16 weeks after surgery, nerve regeneration was assessed using functional, electrophysiologic, histologic, and immunofluorescence analyses. Ultrasonography was used during the survival period to noninvasively evaluate muscle atrophy and reinnervation by measuring cross-sectional muscle area. RESULTS: Surgical angiogenesis of allografts demonstrated significantly improved isometric tetanic force recovery at 12 weeks, compared to allograft alone, which normalized between groups at 16 weeks. Cross-sectional muscle areas showed no differences between groups. Electrophysiology showed superiority of autografts at both time points. No differences were found in histologic analysis, besides a significantly inferior N ratio in allografts at 12 weeks. Immunofluorescent expression of CD34, indicating vascularity, was significantly enhanced in the superficial inferior epigastric artery fascial group compared to allografts at 12 weeks, with highest expression at 16 weeks compared to all groups. CONCLUSION: Surgical angiogenesis with an adipofascial flap to the nerve allograft increases vascularity in the nerve graft, with subsequent improvement of early muscle force recovery, comparable to autografts.

Autogenous Arteriovenous Bundle Implantation Maintains Viability Without Increased Immune Response in Large Porcine Bone Allotransplants.

BACKGROUND: Transplantation of living allogeneic bone segments may permit reconstruction of large defects, particularly if viability is maintained without immunosuppression. Development of a new autogenous osseous blood supply accomplishes this goal in rodent experimental models. This study evaluates potential systemic and local inflammatory responses to this angiogenesis in a large-animal model. METHODS: Vascularized allogeneic tibia segments were transplanted orthotopically into matched tibial defects in Yucatan minipigs. Microvascular anastomoses of bone nutrient artery and vein were supplemented by intramedullary placement of an autogenous arteriovenous (AV) bundle in group 1. Group 2 served as a no-angiogenesis control. A 3-drug immunosuppression regimen was withdrawn after 2 weeks. During the 20-week survival period, periodic leukocyte counts and inflammatory cytokine levels were measured. Thereafter, osteocyte survival was quantified and transplant rejection graded by histologic examination and quantitative real-time polymerase chain reaction of immunologic markers. RESULTS: Both groups developed an initial systemic response, which resolved after 4 to 6 weeks. No differences were seen in blood cytokine levels. Interleukin 2 expression was diminished in group 1 tibiae. As expected, nutrient pedicles had thrombosed without sustained immunosuppression, occluded by intimal hyperplasia. In group 1, angiogenesis from the autogenous AV bundle resulted in significantly less osteonecrosis (P = .04) and fibrosis (P = .02) than group 2 allotransplants. CONCLUSIONS: Systemic immune responses to large-bone allotransplants were not increased by generation of an autogenous osseous blood supply within porcine tibial bone allotransplants. Implanted AV bundles diminished inflammation and fibrosis and improved bone viability when compared to no-angiogenesis controls.

Maximum Isometric Tetanic Force Measurement of the Tibialis Anterior Muscle in the Rat.

Traumatic nerve injuries result in substantial functional loss and segmental nerve defects often necessitate the use of autologous interposition nerve grafts. Due to their limited availability and associated donor side morbidity, many studies in the field of nerve regeneration focus on alternative techniques to bridge a segmental nerve gap. In order to investigate the outcomes of surgical or pharmacological experimental treatment options, the rat sciatic nerve model is often used as a bioassay. There are a variety of outcome measurements used in rat models to determine the extent of nerve regeneration. The maximum output force of the target muscle remains the most relevant outcome for clinical translation of experimental therapies. Isometric force measurement of tetanic muscle contraction has previously been described as a reproducible and valid technique for evaluating motor recovery after nerve injury or repair in both rat and rabbit models. In this video, we will provide a step-by-step instruction of this invaluable procedure for assessment of functional recovery of the tibialis anterior muscle in a rat sciatic nerve defect model using optimized parameters. We will describe the necessary pre-surgical preparations in addition to the surgical approach and dissection of the common peroneal nerve and tibialis anterior muscle tendon. The isometric tetanic force measurement technique will be detailed. Determining the optimal muscle length and stimulus pulse frequency is explained and measuring the maximum tetanic muscle contraction is demonstrated.