The peripheral nerve allograft in the primate immunosuppressed with Cyclosporin A: I. Histologic and electrophysiologic assessment.

Nerve regeneration across peripheral nerve allografts and control autografts in primates immunosuppressed with Cyclosporin A was quantitatively evaluated by electrophysiologic and histologic methods. Twelve cynomolgus monkeys received 3-cm autografts and allografts in contralateral ulnar nerves. They were immunosuppressed with Cyclosporin A at 25 mg/kg per day or placebo vehicle. Morphometric analysis of nerve graft and distal nerve segments was assessed at 1 year after engraftment. Quantitative electrophysiologic studies were performed percutaneously at 6 and 12 months, and compound action potentials were measured directly across the nerve grafts at 1 year. Excellent regeneration was seen across autografts and allografts in Cyclosporin A-treated and placebo-treated recipients.     

The peripheral nerve allograft: an assessment of regeneration across nerve allografts in rats immunosuppressed with cyclosporin A

Lewis rats (RT1(1] were the recipients of 3-cm nerve grafts from syngeneic Lewis donors or allogeneic ACI (RT1a) donors. Microneurosurgical repair of the nerve graft to the transected sciatic nerve of the recipient animal was performed with 10-0 epineurial sutures. Recipients were randomly allocated to cyclosporin A (CsA) immunosuppressed or untreated groups. Cyclosporin A was administered in the minimal effective dosage to prevent nerve allograft rejection across this major histocompatibility disparity (5 mg/kg per day). Nerve regeneration across the nerve grafts was assessed by sciatic function index (SFI) and toe spread index (TSI) determinations serially and by electrophysiologic, histologic, and morphologic assessments 14 weeks after engraftment. Sciatic nerve regeneration across allogeneic nerve grafts in cyclosporin A immunosuppressed recipients was significantly superior compared to the untreated controls (p less than 0.008) and not significantly different from that across the syngeneic control animals.     

Clinical outcome following nerve allograft transplantation

The clinical outcome of seven patients who underwent reconstruction of long upper- and lower-extremity peripheral nerve gaps with interposition peripheral nerve allografts is reported. Patients were selected for transplantation when the nerve gaps exceeded the length that could be reconstructed with available autograft tissue. Before transplantation, cadaveric allografts were harvested and preserved for 7 days in University of Wisconsin Cold Storage Solution at 5 degrees C. In the interim, patients were started on an immunosuppressive regimen consisting of either cyclosporin A or tacrolimus (FK506), azathioprine, and prednisone. Immunosuppression was discontinued 6 months after regeneration across the allograft(s) was evident. Six patients demonstrated return of motor function and sensation in the affected limb, and one patient experienced rejection of the allograft secondary to subtherapeutic immunosuppression. In addition to providing the ability to restore nerve continuity in severe extremity injuries, successful nerve allografting protocols have direct applicability to composite tissue transplantation.     

The peripheral nerve allograft in the primate immunosuppressed with Cyclosporin A: II. Functional evaluation of reinnervated muscle.

Isometric contractile function was evaluated in primates receiving peripheral nerve allografts and autografts. Twelve adult male cynomolgus monkeys received both sural nerve allografts and autografts to the ulnar nerve in opposite forearms. Half the animals received Cyclosporin A (CsA) immunosuppression (25 mg/kg per day); the remaining animals received placebo. One year following nerve engraftment, isometric contractile muscle function was evaluated in reinnervated abductor digiti quinti and intact abductor pollicis brevis muscles. Maximal twitch tension (Pt), tetanic tension (P(o)), time to peak tension (tpt), rate of rise of twitch tension (DP/dt), and muscle fatigue were evaluated at optimal muscle length (L(o)). All reinnervated muscles distal to nerve autografts and allografts in both Cyclosporin A-immunosuppressed and placebo-treated animals generated equivalent maximal twitch tension, tetanic tension, and time to peak tension, with no significant difference between groups (p > 0.05 by ANOVA). There was a tendency toward increased muscle fatiguability in Cyclosporin A-treated animals (p > 0.05). However, the rate of rise of twitch tension was significantly faster in the reinnervated and intact muscles of Cyclosporin A-treated primates (p < 0.05). Evidence of excellent functional reinnervation across nerve allografts and autografts similar to that seen in histologic and electrophysiologic studies was noted. Cyclosporin A immunosuppression did not significantly enhance recovery of muscle function distal to nerve allografts in this model.     

Atrial natriuretic factor (ANF) levels are elevated in rats bearing rejecting heart-lung allografts

Intra-abdominal heart-lung grafts were transplanted into 8 rats across a major histocompatibility barrier. Four of the 8 rats were treated with Cyclosporin A (CsA) to prevent rejection. Atrial natriuretic factor (ANF) levels measured 6 days after transplantation revealed a significantly (p less than .005) higher mean ANF concentration in rats bearing a rejecting heart-lung allograft (642 +/- 148.0 pg/ml) compared to rats bearing a heart-lung allograft not undergoing rejection (200.8 +/- 13.07 pg/ml). ANF might be a useful noninvasive marker in the diagnosis of rejection in heart and heart-lung transplants.     

Anti-CD40 ligand antibody permits regeneration through peripheral nerve allografts in a nonhuman primate model

Systemic immunosuppression is typically required to prevent allograft rejection. Antibody-based therapies that induce immune unresponsiveness represent an appealing alternative to nonspecific immunosuppression, which is often associated with significant morbidity. In mice, successful prevention of nerve allograft rejection has been demonstrated through interference with the CD40/CD40 ligand interaction. This study investigated the effectiveness of anti-CD40 ligand monoclonal antibody as single-agent therapy in preventing rejection and supporting nerve regeneration across long nerve allografts in nonhuman primates. Twelve outbred cynomolgus macaques were arranged into six genetically mismatched pairs, with each animal receiving a 5-cm ulnar nerve allograft in the right arm and a 5-cm autograft in the left arm. Mixed lymphocyte reaction assays were used to assess resulting immune unresponsiveness. Treated animals (n = 10) received anti-CD40 ligand monoclonal antibody 10 mg/kg one time, locally applied, and 20 mg/kg systemically on postoperative days 0, 1, 3, 10, 18, and 28, and then monthly. Untreated animals (n = 2) served as the untreated controls. At 4 or 6 months after transplantation, nerves were harvested for histological analysis. Four treated animals underwent an additional challenge after cessation of anti-CD40 ligand monoclonal antibody therapy and nerve graft harvests. Autogenous and allogeneic skin and nerve inlay grafting was performed to assess the permanence of immune unresponsiveness induced by anti-CD40 ligand monoclonal antibody. Animals that received anti-CD40 ligand monoclonal antibody demonstrated robust regeneration across nerve allografts, similar to that seen in the autograft control in the contralateral arm. The histomorphometric analysis of allografts in the untreated animals demonstrated significantly worse measurements compared with their matched autograft controls. Animals that received anti-CD40 ligand monoclonal antibody with concomitant skin allografts had virtually no evidence of nerve regeneration through allografts. Allogeneic skin and nerve allografts applied 2 to 12 months after withdrawal of anti-CD40 ligand monoclonal antibody therapy were consistently rejected. This study demonstrates that anti-CD40 ligand monoclonal antibody prevents rejection and allows regeneration of peripheral nerve allografts in nonhuman primates. The effect of anti-CD40 ligand monoclonal antibody appears to be transient, however, with restoration of immunocompetence shortly after withdrawal of therapy.     

Disseminated coccidioidomycosis in a heart transplant recipient

A cardiac transplant patient developed disseminated coccidioidomycosis shortly after transplantation and institution of immunosuppressive therapy. The patient was maintained on intravenous and intrathecal amphotericin B for 19 months, but when therapy was discontinued, the disease relapsed and he died. At autopsy the cardiac allograft was without signs of rejection, but the patient had coccidioidomycotic lesions in multiple organs. There is an increasing number of reports of disseminated coccidioidomycosis in immunocompromised patients, especially those who receive steroids or immunosuppressive therapy. Coccidioidomycosis may represent a severe complication in the transplant patient.     

The peripheral nerve allograft: a dose-response curve in the rat immunosuppressed with cyclosporin A

The potential use of peripheral nerve allografts would significantly improve the reconstructive potential for patients with major peripheral nerve deficits. This study evaluated the response of the nerve allograft recipient treated with varying dosages of cyclosporin A (CsA) to determine the minimal effective dosage necessary to prevent nerve graft rejection. Lewis rats (RT1l) were the recipients of syngeneic nerve grafts from identical Lewis donors or allogeneic nerve grafts from ACI (RT1a) donors. Nerve grafts were inlaid next to the intact sciatic nerve of the recipient. The immunologic responsiveness of the recipient animal's lymphocytes to a donor-specific antigenic challenge was assessed by the mixed lymphocyte reaction (MLR). In addition, nerve grafts were evaluated histologically. Animals were monitored for cyclosporin A toxicity. It was found that cyclosporin A (5 mg/kg per day) was effective in rendering the recipient animals unresponsive by mixed lymphocyte reaction at 10, 20, and 40 days after engraftment. This dosage was similarly effective in preventing histologic changes characteristic of nerve allograft rejection. This dosage regimen was nontoxic to the animals. Our study ascertained a minimal nontoxic dosage of cyclosporin A that effectively prevented nerve allograft rejection across a major histocompatibility disparity in rats.     

神经生长因子与冻干异体神经桥接大鼠神经缺损的研究

实验采用冻干处理的异体神经与外源性神经生长因子（ＮＧＦ）结合来桥接大鼠的坐骨神经１．０ｃｍ的缺损。用雄性Ｗｉｓｔａｒ大鼠进行的四组实验结果表明：冻干处理的异体神经可降低其抗原性,但处理后并不损害雪旺氏细胞（ＳＣ）基底膜的完整性,在移植后可能成为轴突再生的通道和支架;外源性ＮＧＦ与冻干神经结合形成的复合体,可为神经的再生提供一个较好的微环境,具有成为理想桥接材料的可能性     

Profiling circulating microRNA expression in a mouse model of nerve allotransplantation

Background

The lack of noninvasive biomarkers of rejection remains a challenge in the accurate monitoring of deeply buried nerve allografts and precludes optimization of therapeutic intervention. This study aimed to establish the expression profile of circulating microRNAs (miRNAs) during nerve allotransplantation with or without immunosuppression.

Results

Balb/c mice were randomized into 3 experimental groups, that is, (1) untreated isograft (Balb/c → Balb/c), (2) untreated allograft (C57BL/6 → Balb/c), and (3) allograft (C57BL/6 → Balb/c) with FK506 immunosuppression. A 1-cm Balb/c or C57BL/6 donor sciatic nerve graft was transplanted into sciatic nerve gaps created in recipient mice. At 1, 3, 7, 10, and 14 d after nerve transplantation, nerve grafts, whole blood, and sera were obtained for miRNA expression analysis with an miRNA array and subsequent validation with quantitative real-time PCR (qRT-PCR). Three circulating miRNAs (miR-320, miR-762, and miR-423-5p) were identified in the whole blood and serum of the mice receiving an allograft with FK506 immunosuppression, within 2 weeks after nerve allotransplantation. However, these 3 circulating miRNAs were not expressed in the nerve grafts. The expression of all these 3 upregulated circulating miRNAs significantly decreased at 2, 4, and 6 d after discontinuation of FK506 immunosuppression. In the nerve graft, miR-125-3b and miR-672 were significantly upregulated in the mice that received an allograft with FK506 only at 7 d after nerve allotransplantation.

Conclusions

We identified the circulating miR-320, miR-762, and miR-423-5p as potential biomarkers for monitoring the immunosuppression status of the nerve allograft. However, further research is required to investigate the mechanism behind the dysregulation of these markers and to evaluate their prognostic value in nerve allotransplantation.     

The role of cyclosporin in prolonging survival in vascularized bone allografts

It is known that experimental vascularized bone allografts are subject to host rejection. To be useful clinically, this rejection response would need to be controlled. Cyclosporin is a potent immunosuppressant whose precise role in vascularized bone allograft transplantation has not been established. Using a proven reliable vascularized knee allograft model in inbred rats, cyclosporin was used postoperatively both continuously and short term (14 days after transplant) at 10 mg/kg per day as recipient treatment. Across a strong histocompatibility barrier, continuous cyclosporin was required for long-term graft survival. Short-term therapy delayed rejection for 4 to 6 weeks. However, across a weak histocompatibility barrier, short-term therapy was as effective as continuous therapy in achieving long-term graft survival. The implication is that a limited course of cyclosporin may be clinically successful in sustaining vascularized bone allograft survival, provided the genetic disparity between graft and host has been minimized by genetic matching techniques.     

Long-term survival of transplanted induced pluripotent stem cell-derived neurospheres with nerve conduit into sciatic nerve defects in immunosuppressed mice

Since the advent of induced pluripotent stem cells (iPSCs), clinical trials using iPSC-based cell transplantation therapy have been performed in various fields of regenerative medicine. We previously demonstrated that the transplantation of mouse iPSC-derived neurospheres containing neural stem/progenitor cells with bioabsorbable nerve conduits promoted nerve regeneration in the long term in murine sciatic nerve defect models. However, it remains unclear how long the grafted iPSC-derived neurospheres survived and worked after implantation. In this study, the long-term survival of the transplanted mouse iPSC-derived neurospheres with nerve conduits was evaluated in high-immunosuppressed or non-immunosuppressed mice using in vivo imaging for the development of iPSC-based cell therapy for peripheral nerve injury. Complete 5-mm long defects were created in the sciatic nerves of immunosuppressed and non-immunosuppressed mice and reconstructed using nerve conduits coated with iPSC-derived neurospheres labeled with ffLuc. The survival of mouse iPSC-derived neurospheres on nerve conduits was monitored using in vivo imaging. The transplanted iPSC-derived neurospheres with nerve conduits survived for 365 days after transplantation in the immunosuppressed allograft models, but only survived for at least 14 days in non-immunosuppressed allograft models. This is the first study to find the longest survival rate of stem cells with nerve conduits transplanted into the peripheral nerve defects using in vivo imaging and demonstrates the differences in graft survival rate between the immunosuppressed allograft model and immune responsive allograft model. In the future, if iPSC-derived neurospheres are successfully transplanted into peripheral nerve defects with nerve conduits using iPSC stock cells without eliciting an immune response, axonal regeneration will be induced due to the longstanding supportive effect of grafted cells on direct remyelination and/or secretion of trophic factors.     

Blood cyclosporin concentrations and renal allograft dysfunction.

Forty nine renal allograft recipients taking oral cyclosporin suffered 76 episodes of renal dysfunction within six months of transplantation. These episodes were diagnosed as graft rejection or cyclosporin induced nephrotoxicity on the basis of histological findings in allograft biopsy specimens and the response to treatment. Mean predose blood cyclosporin concentrations measured by radioimmunoassay during the week before the onset of renal dysfunction were significantly higher when the cause was cyclosporin toxicity rather than graft rejection (392 v 741 nmol/l (471 v 891 ng/ml). During this period there was a significant association between both the frequency of measurements above 666 nmol/l (800 ng/ml) and the diagnosis of toxicity and the frequency of measurements below 333 nmol/l (400 ng/ml) and the diagnosis of allograft rejection. Cyclosporin measurements made at the time of biopsy and reference to the highest or lowest concentrations measured during the week preceding biopsy were of less value in distinguishing between the two groups. Despite lacking specificity for the parent compound, the radioimmunoassay used produced results which were of clinical value in optimising cyclosporin treatment.     

Brachial plexus injury.

A 28-year-old man shot himself in the left posterior triangle of the neck with a shotgun. At the initial operation secondary repair of the resultant brachial plexus injury was decided upon in view of the difficulty in assessing lesions in continuity at this point after injury. The patient had total brachial plexus palsy. Nine weeks after the injury sensory and motor function were returning and the only element of the brachial plexus not showing evidence of nerve fibre continuity was the musculocutaneous nerve. Sural nerve autografts were sutured between the trimmed proximal and distal stumps of this nerve. By 4 months after the injury there was further improvement in both sensory and motor function, and by 18 months there was sensation in the autonomous zones of both median and ulnar nerves and good return of muscle power.     

Diabetogenic effect of cyclosporin.

A young woman given a renal allograft for polycystic kidney disease developed insulin dependent diabetes mellitus 25 days after transplantation. There was no family history of diabetes, plasma glucose concentrations had been normal at presentation and on five subsequent occasions, and at no time were islet cell antibodies detectable. Plasma C peptide concentrations, however, were greatly suppressed after transplantation and remained so for up to six months. The immunosuppressive regimen had included cyclosporin A, which had been difficult to regulate and caused definite signs of toxicity in the patient. By virtue of its reported toxicity for beta cells and the reversal of the diabetes several months after the dose was reduced cyclosporin was incriminated as the probable causative agent. Dose related beta cell toxicity of cyclosporin A may be a risk in recipients of this drug and warrants careful monitoring of drug and glucose concentrations.     

Botulinum toxin: a treatment for facial asymmetry caused by facial nerve paralysis

Injury to the frontal or other facial nerve branches can result in an asymmetry that can be very distressful to both patient and surgeon. This is especially true following cosmetic procedures such as rhytidectomy. We propose a means to create temporary symmetry while awaiting the possible return of nerve function. Botulinum neurotoxin causes a muscle paralysis lasting for approximately 3 months, and it is well established as the preferred treatment for blepharospasm. A case is presented in which botulinum toxin type A was injected into the opposite functioning frontalis muscle of a patient with unilateral frontal nerve paralysis. The patient experienced satisfactory relief of the asymmetry caused by onesided forehead wrinkling and brow elevation. Botulinum toxin therapy should be considered for both temporary and permanent facial asymmetries due to facial nerve paralysis as well as spasm.     

Ocular fixation with quadriceps tendon allograft

Management of difficult strabismus, such as strabismus fixus and paralytic strabismus, in order to maintain the alignment is complicated. There are many surgical approaches described in the current literature, together with notes on the materials used to stabilize these deformities. We present a new surgical approach using quadriceps tendon allograft for the correction of difficult strabismus such as strabismus fixus and paralytic strabismus. Our idea for using deep frozen quadriceps tendons developed from the fact that this is the most stable and strongest tendon in the whole body. Six patients with strabismus fixus (n = 1), paralytic strabismus secondary to extraocular muscle damage (n = 1) and combined cranial nerve palsy (n = 4) were operated on using a strip of deep frozen quadriceps tendon allograft suturedonto both the globe and the periosteum. The mean age of the patients was 44.33years. (range 17–71 years) All the patients were followed up for six months. The mean preoperative deviation in the six cases was 60 prism diopters(PD) (range 30–123 PD) The mean change in horizontal alignment at 1 month, 3 months and 6 months postoperatively was 54PD, 53 PD and 49.16 PD respectively. We had only one case of under correction. This may possible be due to the remaining function of the antagonist and/or the fact that we had used the distal end of the quadriceps tendon. When the patient (patient No. 5) who had 2 operations was excluded, the mean change in horizontal alignment was 48.6 PD,47.4PD and 43.6 PD. Post-operatively there was no infection or any other complications. According to our study of existing literature, these cases of deep frozen quadriceps tendon allograft application for the indications mentioned above are the first reported cases of its type. We conclude that ocular fixation with quadriceps tendon to the periosteum is a safe and effective option for the management of difficult strabismus. Further research on a larger cohort of patients and longer follow-up time are needed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cyclosporin A as prophylaxis against graft-versus-host disease in 36 patients.

Oral cyclosporin A was used as prophylaxis against graft-versus-host disease in (a) 31 patients with acute leukaemia or aplastic anaemia given transplants of HLA-matched bone marrow and (b) five patients with inborn errors of metabolism given transplants of haplotype-identical (parental) bone marrow. Twenty-six patients survived longer than two months after the operation. Despite the cyclosporin A, 31 patients (86%) suffered an acute form of graft-versus-host disease and 22 (61%) a chronic form. Nevertheless, the disease was usually treatable with immunosuppressive agents and caused the death of only one patient. Cyclosporin A caused renal toxicity in all cases; occasionally this was associated with a "capillary leak" syndrome, fatal in two patients. In children hypertension, fits, and fluid retention were common side effects. Blood concentrations of cyclosporin A correlated with blood urea values and blood pressure but did not predict the occurrence of graft-versus-host disease. Four different dose schedules were used to find the optimum way to administer this drug. Oral cyclosporin A is extremely effective at reducing the severity of graft-versus-host disease, but prevention of the disease is limited by toxicity of the drug and variable absorption. Better results might be achieved with parenteral administration or by using the drug in combination with other methods.     

Intraocular transplants of a human gastrinoma in immuno-suppressed rats: morphological, chromatographic and functional studies

Tissue pieces of a metastatic human gastrinoma (ultrastructural Type II) were successfully transplanted to the anterior eye-chamber of rats immunosuppressed with Cyclosporin A. Immunocytochemical investigation of the transplants showed evidence for preserved endocrine activity of tumour cells with immunoreactivity towards the C-terminal of the gastrin/cholecystokinin molecule. Studies of gastric acid secretion in tumour-bearing rats and sham-operated controls with chronic gastric fistulas showed that the basal acid output did not differ between the groups during 3 weeks of study. However, the stimulated gastric acid secretion decreased after 5 days in both groups to remain significantly depressed throughout the study, an effect probably due to Cyclosporin A treatment of the groups. The concentration of immunoreactive gastrin in plasma from rats with tumours in oculo was 5 times higher than in sham-operated rats. Gastrin-34 was the major immunoreactive component in both patient serum and rat plasma. An immunoreactive fraction corresponding to component I was found in the patient serum, but not in the rat plasma, although present in the chamber fluid. Components corresponding to gastrin-17 were found both in the patient serum and in the rat plasma. The chromatographic pattern of the tumour was similar to that in rat chamber fluid. The dominating component corresponded to gastrin-17, while gastrin-34 represented the quantitatively smaller component. Gastrin-34 was, however, relatively more abundant in the tumour extract than in the chamber fluid. The study also indicates that a gastrin-producing tumour transplanted in oculo in immunosuppressed rats may increase the rat plasma concentration of the same molecular forms of gastrin as seen in the clinical situation.     

Ultrastructure of a sciatic nerve allograft preserved at an ultralow temperature

In the experiment, performed on dogs ultrastructure of the ischiatic nerve allograft, preserved in liquid nitrogen (-196 degrees) has been studied. In 7 days after the operation in the transplanted piece of the nerve trunk certain phenomena, similar to the Wallerian degeneration are noted, while viability of neurilemma cells is preserved. Processes of second degeneration are mainly completed by the end of the first month after the operation. During this time active regeneration of nervous fibers is noted; it is accompanied with neutralization of the graft. The latter is mostly expressed in 6 months after the operation. The dynamics studied on ultramicroscopic rearrangements of the cryopreserved graft proves the previously obtained data on possibility of its use in clinics for substitution of defects in peripheral nerves.