Olawale A.R. Sulaiman

Experimental strategies to promote functional recovery after peripheral nerve injuries

Abstract  The capacity of Schwann cells (SCs) in the peripheral nervous system to support axonal regeneration, in contrast to the oligodendrocytes in the central nervous system, has led to the misconception that peripheral nerve regeneration always restores function. Here, we consider how prolonged periods of time that injured neurons remain without targets during axonal regeneration (chronic axotomy) and that SCs in the distal nerve stumps remain chronically denervated (chronic denervation) progressively reduce the number of motoneurons that regenerate their axons. We demonstrate the effectiveness of low‐dose, brain‐derived neurotrophic and glial‐derived neurotrophic factors to counteract the effects of chronic axotomy in promoting axonal regeneration. High‐dose brain‐derived neurotrophic factor (BDNF) on the other hand, acting through the p75 receptor, inhibits axonal regeneration and may be a factor in stopping regenerating axons from forming neuromuscular connections in skeletal muscle. The immunophilin, FK506, is also effective in promoting axonal regeneration after chronic axotomy. Chronic denervation of SCs (>1 month) severely deters axonal regeneration, although the few motor axons that do regenerate to reinnervate muscles become myelinated and form enlarged motor units in the reinnervated muscles. We found that in vitro incubation of chronically denervated SCs with transforming growth factor‐β re‐established their growth‐supportive phenotype in vivo, consistent with the idea that the interaction between invading macrophages and denervated SCs during Wallerian degeneration is essential to sustain axonal regeneration by promoting the growth‐supportive SC phenotype. Finally, we consider the effectiveness of a brief period of 20 Hz electrical stimulation in promoting the regeneration of axons across the surgical gap after nerve repair.

Effects of short- and long-term Schwann cell denervation on peripheral nerve regeneration, myelination, and size

Poor functional recovery after peripheral nerve injury has been generally attributed to inability of denervated muscles to accept reinnervation and recover from denervation atrophy. However, deterioration of the Schwann cell environment may play a more vital role. This study was undertaken to evaluate the effects of chronic denervation on the capacity of Schwann cells in the distal nerve stump to support axonal regeneration and to remyelinate regenerated axons. We used a delayed cross‐suture anastomosis technique in which the common peroneal (CP) nerve in the rat was denervated for 0–24 weeks before cross‐suture of the freshly axotomized tibial (TIB) and chronically denervated CP nerve stumps. Motor neurons were backlabeled with either fluoro‐ruby or fluorogold 12 months later, to identify and count TIB motor neurons that regenerated axons into chronically denervated CP nerve stumps. Number, size, and myelination of regenerated sensory and motor axons were determined using light and electron microscopy. We found that short‐term denervation of ≤4weeks did not affect axonal regeneration but more prolonged denervation profoundly reduced the numbers of backlabeled motor neurons and axons in the distal nerve stump. Yet, atrophic Schwann cells retained their capacity to remyelinate regenerated axons. In fact, the axons were larger and well myelinated by long‐term chronically denervated Schwann cells. These findings demonstrate a progressive inability of chronically denervated Schwann cells to support axonal regeneration and yet a sustained capacity to remyelinate the axons which do regenerate. Thus, axonal interaction can effectively switch the nonmyelinating phenotype of atrophic Schwann cells back into the myelinating phenotype. GLIA 32:234–246, 2000.

A decline in glial cell-line-derived neurotrophic factor expression is associated with impaired regeneration after long-term Schwann cell denervation.

In the peripheral nervous system, regeneration of motor and sensory axons into chronically denervated distal nerve segments is impaired compared to regeneration into acutely denervated nerves. In order to find possible causes for this phenomenon we examined the changes in the expression pattern of the glial cell-line-derived neurotrophic factor (GDNF) family of growth factors and their receptors in chronically denervated rat sciatic nerves as a function of time with or without regeneration. Among the GDNF family of growth factors, only GDNF mRNA expression was rapidly upregulated in Schwann cells as early as 48 h after denervation. This upregulation peaked at 1 week and then declined to minimal levels by 6 months of denervation. The changes in the protein expression paralleled the changes in the expression of the GDNF mRNA. The mRNAs for receptors GFRalpha-1 and GFRalpha-2 were upregulated only after maximal GDNF upregulation and remained elevated as late as 6 months. There were no significant changes in the expression of GFRalpha-3 or the tyrosine kinase coreceptor, RET. When we examined the expression of GDNF in a delayed regeneration paradigm, there was no upregulation in the distal chronically denervated tibial nerve even when the freshly axotomized peroneal branch of the sciatic nerve was sutured to the distal tibial nerve. This study suggests that one of the reasons for impaired regeneration into chronically denervated peripheral nerves may be the inability of Schwann cells to maintain important trophic support for both motor and sensory neurons.

FK506 increases peripheral nerve regeneration after chronic axotomy but not after chronic schwann cell denervation.

Poor functional recovery after peripheral nerve injury is attributable, at least in part, to chronic motoneuron axotomy and chronic Schwann cell (SC) denervation. While FK506 has been shown to accelerate the rate of nerve regeneration following a sciatic nerve crush or immediate nerve repair, for clinical application, it is important to determine whether the drug is effective after chronic nerve injuries. Two models were employed in the same adult rats using cross-sutures: chronic axotomy and chronic denervation of SCs. For chronic axotomy, a chronically (2 months) injured proximal tibial (TIB) was sutured to a freshly cut common peroneal (CP) nerve. For chronic denervation, a chronically (2 months) injured distal CP nerve was sutured to a freshly cut TIB nerve. Rats were given subcutaneous injections of FK506 or saline (5 mg/kg/day) for 3 weeks. In the chronic axotomy model, FK506 doubled the number of regenerated motoneurons identified by retrograde labeling (from 205 to 414 TIB motoneurons) and increased the numbers of myelinated axons (from 57 to 93 per 1000 microm2) and their myelin sheath thicknesses (from 0.42 to 0.78 microm) in the distal nerve stump. In contrast, after chronic denervation, FK506 did not improve the reduced capacity of SCs to support axonal regeneration. Taken together, the results suggest that FK506 acts directly on the neuron (as opposed to the denervated distal nerve stump) to accelerate and promote axonal regeneration of neurons whose regenerative capacity is significantly reduced by chronic axotomy.

Role of chronic Schwann cell denervation in poor functional recovery after nerve injuries and experimental strategies to combat it.

OBJECTIVETo present our data about the role of chronic denervation (CD) of the distal nerve stumps as compared with muscle denervation atrophy and experimental strategies to promote better functional recovery. METHODSA rat model of nerve injury and repair was used. The common peroneal branch of the sciatic nerve was subjected to 0 to 24 weeks of CD before cross-suture with the tibial motoneurons. Our outcome measures included the numbers of motoneurons that regenerated their axons and the numbers that reinnervated muscle targets (motor units). To overcome the effects of CD, we used subcutaneous injection of FK506 and in vitro reactivation of Schwann cells that had been subjected to 24 weeks of CD with transforming growth factor β. RESULTSNumbers of regenerated motoneurons and reinnervated motor units decreased as a function of duration of CD. However, axons that regenerated through the distal nerve stumps reinnervated the muscle targets and even formed enlarged motor unit size regardless of the duration of CD. FK506 doubled the numbers of tibial motoneurons that regenerated their axons into the common peroneal nerve even after delayed repair. Reactivation of chronically denervated Schwann cells with transforming growth factor β significantly increased their capacity to support axonal regeneration. CONCLUSIONCD of the distal nerve stumps is the primary factor that results in poor axonal regeneration and subsequently poor functional recovery. Acceleration of the rate of axonal regeneration and/or reactivation of Schwann cells of the distal nerve stumps are effective experimental strategies to promote axonal regeneration and functional recovery.

Transforming growth factor-β and forskolin attenuate the adverse effects of long-term Schwann cell denervation on peripheral nerve regeneration in vivo

Transforming growth factor‐β (TGF‐β) plays a central role in the regulation of Schwann cell (SC) proliferation and differentiation and is essential for the neurotrophic effects of several neurotrophic factors (reviewed by Unsicker and Krieglstein, 2000 ; Unsicker and Strelau, 2000 ). However, its role in peripheral nerve regeneration in vivo is not yet understood. Our studies were carried out to characterize (1) the effects of duration of regeneration, and chronic SC denervation on the number of tibial (TIB) motor neurons that regenerated axons over a fixed distance (25 mm into distal common peroneal [CP] nerve stumps), and (2) the effect of in vitro incubation of 6‐month chronically denervated sciatic nerve explants with TGF‐β and forskolin on their capacity to support axonal regeneration in vivo. TIB–CP cross‐suture in Silastic tubing was used, and regeneration into 0–24‐week chronically denervated CP stumps was allowed for either 1.5 or 3 months. Chronically denervated rat sciatic nerve explants (3 × 3 mm2) were incubated in vitro with either DMEM and 15% fetal calf serum (D‐15) plus TGF‐β/forskolin or D‐15 alone for 48 h and placed into a 10‐mm Silastic tube that bridged the proximal and distal nerve stumps of a freshly cut TIB nerve. The number of tibial motor neurons that regenerated axons through the explants and 25 mm into the distal nerve stump after 6 months, and TIB regeneration into the CP nerve stumps, were assessed using retrograde tracers, fluorogold, or fluororuby. We found that all tibial motor neurons regenerate their axons 25 mm into 0–4‐week denervated CP nerve stumps after a regeneration period of 3 months. Reducing regeneration time to 1.5 months and chronic denervation, reduced the number of motor neurons that regenerated axons over 25 mm. Exposure of 6‐month denervated nerve explants to TGF‐β/forskolin increased the number of motor neurons that regenerated through them from 258 ± 13; mean ± SE to 442 ± 22. Hence, acute treatment of atrophic SC with TGF‐β can reactivate the growth‐permissive SC phenotype to support axonal regeneration. GLIA 37:206–218, 2002.

Chronic Schwann Cell Denervation and the Presence of a Sensory Nerve Reduce Motor Axonal Regeneration

Motor axonal regeneration is compromised by chronic distal nerve stump denervation, induced by delayed repair or prolonged regeneration distance, suggesting that the pathway for regeneration is progressively impaired with time and/or distance. In the present experiments, we tested the impacts of (i) chronic distal sensory nerve stump denervation on axonal regeneration and (ii) sensory or motor innervation of a nerve graft on the ability of motoneurons to regenerate their axons from the opposite end of the graft. Using the motor and sensory branches of rat femoral nerve and application of neuroanatomical tracers, we evaluated the numbers of regenerated femoral motoneurons and nerve fibers when motoneurons regenerated (i) into freshly cut and 2-month chronically denervated distal sensory nerve stump, (ii) alone into a 4-cm-long distally ligated sensory autograft (MGL) and, (iii) concurrently as sensory (MGS) or motor (MGM) nerves regenerated into the same autograft from the opposite end. We found that all (315 +/- 24: mean +/- SE) the femoral motoneurons regenerated into a freshly cut distal sensory nerve stump as compared to 254 +/- 20 after 2 months of chronic denervation. Under the MGL condition, 151 +/- 5 motoneurons regenerated, which was not significantly different from the MGM group (134 +/- 13) but was significantly reduced to 99 +/- 2 in the MGS group (P < 0.05). The number of regenerated nerve fibers was 1522 +/- 81 in the MGL group, 888 +/- 18 in the MGM group, and 516 +/- 44 in the MGS group, although the high number of nerve fibers in the MGL group was due partly to the elaboration of multiple sprouts. Nerve fiber number and myelination were reduced in the MGS group and increased in the MGM group. These results demonstrate that both chronic denervation and the presence of sensory nerve axons reduced desired motor axonal regeneration into sensory pathways. A common mechanism may involve reduced responsiveness of sensory Schwann cells within the nerve graft or chronically denervated distal nerve stump to regenerating motor axons. The findings confirm that motor regeneration is optimized by avoiding even short-term denervation. They also imply that repairing pure motor nerves (without their cutaneous sensory components) to distal nerve stumps should be considered clinically when motor recovery is the main desired outcome.

Nerve transfer surgery for adult brachial plexus injury: A 10-year experience at Louisiana State University

OBJECTIVETo review the clinical outcomes in our patients who have undergone nerve transfer operations for brachial plexus reconstruction at the Louisiana State University (LSU) over a 10-year period. A secondary objective is to compare clinical outcomes in patients who had only nerve transfer operations as compared with patients whose nerve transfers were supplemented with direct repair of brachial plexus elements. METHODSRetrospective review of the medical records, imaging, and electrodiagnostic studies (electromyographic and nerve conduction studies) of patients with brachial plexus injuries who underwent nerve transfer operations at LSU over a period of 10 years. RESULTSA total of 81 patients were treated between 1995 to 2005 at the LSU Health Sciences Center; 7 of these patients were lost to follow-up, leaving 74 patients, with an average follow-up of 3.5 years, for review. We evaluated recovery of elbow flexion and shoulder abduction. Ninety percent of patients with medial pectoral to musculocutaneous nerve transfers recovered to LSU grade 2 (Medical Research Council grade 3), and 60% of those patients with intercostal to musculocutaneous nerve transfer regained similar strength in elbow flexion. Shoulder abduction recovery to LSU grade 2 (Medical Research Council grade 3) after spinal accessory to suprascapular and/or thoracodorsal to axillary nerve transfer, was 95% and 36%, respectively. There was a tendency for better motor recovery when nerve transfer operations were combined with direct repair of plexus elements. CONCLUSIONNerve transfers for repair of brachial plexus injuries result in excellent recovery of elbow and shoulder functions. Patients who had direct repair of brachial plexus elements in addition to nerve transfers tended to do better than those who had only nerve transfer operations.OBJECTIVE To review the clinical outcomes in our patients who have undergone nerve transfer operations for brachial plexus reconstruction at the Louisiana State University (LSU) over a 10-year period. A secondary objective is to compare clinical outcomes in patients who had only nerve transfer operations as compared with patients whose nerve transfers were supplemented with direct repair of brachial plexus elements. METHODS Retrospective review of the medical records, imaging, and electrodiagnostic studies (electromyographic and nerve conduction studies) of patients with brachial plexus injuries who underwent nerve transfer operations at LSU over a period of 10 years. RESULTS A total of 81 patients were treated between 1995 to 2005 at the LSU Health Sciences Center; 7 of these patients were lost to follow-up, leaving 74 patients, with an average follow-up of 3.5 years, for review. We evaluated recovery of elbow flexion and shoulder abduction. Ninety percent of patients with medial pectoral to musculocutaneous nerve transfers recovered to LSU grade 2 (Medical Research Council grade 3), and 60% of those patients with intercostal to musculocutaneous nerve transfer regained similar strength in elbow flexion. Shoulder abduction recovery to LSU grade 2 (Medical Research Council grade 3) after spinal accessory to suprascapular and/or thoracodorsal to axillary nerve transfer, was 95% and 36%, respectively. There was a tendency for better motor recovery when nerve transfer operations were combined with direct repair of plexus elements. CONCLUSION Nerve transfers for repair of brachial plexus injuries result in excellent recovery of elbow and shoulder functions. Patients who had direct repair of brachial plexus elements in addition to nerve transfers tended to do better than those who had only nerve transfer operations.