Neil Tyreman

The Basis for Diminished Functional Recovery after Delayed Peripheral Nerve Repair

The postsurgical period during which neurons remain without target connections (chronic axotomy) and distal nerve stumps and target muscles are denervated (chronic denervation) deleteriously affects functional recovery. An autologous nerve graft and cross-suture paradigm in Sprague Dawley rats was used to systematically and independently control time of motoneuron axotomy, denervation of distal nerve sheaths, and muscle denervation to determine relative contributions of each factor to recovery failure. Tibial (TIB) nerve was cross-sutured to common peroneal (CP) nerve via a contralateral 15 mm nerve autograft to reinnervate the tibialis anterior (TA) muscle immediately or after prolonging TIB axotomy, CP autograft denervation, or TA muscle denervation. Numbers of motoneurons that reinnervated TA muscle declined exponentially from 99±15 to asymptotic mean (±SE) values of 35 ± 1, 41 ± 10, and 13 ± 5, respectively. Enlarged reinnervated motor units fully compensated for reduced motoneuron numbers after prolonged axotomy and autograft denervation, but the maximal threefold enlargement did not compensate for the severe loss of regenerating nerves through chronically denervated nerve stumps and for failure of reinnervated muscle fibers to recover from denervation atrophy. Muscle force, weight, and cross-sectional area declined. Our results demonstrate that chronic denervation of the distal stump plays a key role in reduced nerve regeneration, but the denervated muscle is also a contributing factor. That chronic Schwann cell denervation within the nerve autograft reduced regeneration less than after the denervation of both CP nerve stump and TA muscle, argues that chronic muscle denervation negatively impacts nerve regeneration.

Preferential motor unit loss in the SOD1G93A transgenic mouse model of amyotrophic lateral sclerosis

The present study investigated motor unit (MU) loss in a murine model of familial amyotrophic lateral sclerosis (ALS). The fast‐twitch tibialis anterior (TA) and medial gastrocnemius (MG) muscles of transgenic SOD1G93A and SOD1WT mice were studied during the presymptomatic phase of disease progression at 60 days of age. Whole muscle maximum isometric twitch and tetanic forces were 80% lower (P < 0.01) in the TA muscles of SOD1G93A compared to SOD1WT mice. Enumeration of total MU numbers within TA muscles showed a 60% reduction (P < 0.01) within SOD1G93A mice (38 ± 7) compared with SOD1WT controls (95 ± 12); this was attributed to a lower proportion of the most forceful fast‐fatigable (FF) MU in SOD1G93A mice, as seen by a significant (P < 0.01) leftward shift in the cumulative frequency histogram of single MU forces. Similar patterns of MU loss and corresponding decreases in isometric twitch force were observed in the MG. Immunocytochemical analyses of the entire cross‐sectional area (CSA) of serial sections of TA muscles stained with anti‐neural cell adhesion molecule (NCAM) and various monoclonal antibodies for myosin heavy chain (MHC) isoforms showed respective 65% (P < 0.01) and 28% (P < 0.05) decreases in the number of innervated IIB and IID/X muscle fibres in SOD1G93A, which paralleled the 60% decrease (P < 0.01) in the force generating capacity of individual fibres. The loss of fast MUs was partially compensated by activity‐dependent fast‐to‐slower fibre type transitions, as determined by increases (P < 0.04) in the CSA and proportion of IIA fibres (from 4% to 14%) and IID/X fibres (from 31% to 39%), and decreases (P < 0.001) in the CSA and proportion of type IIB fibres (from 65% to 44%). We conclude that preferential loss of IIB fibres is incomplete at 60 days of age, and is consistent with a selective albeit gradual loss of FF MUs that is not fully compensated by sprouting of the remaining motoneurons that innervate type IIA or IID/X muscle fibres. Our findings indicate that disease progression in fast‐twitch muscles of SOD1G93A mice involves parallel processes: (1) gradual selective motor axon die‐back of the FF motor units that contain large type IIB muscle fibres, and of fatigue‐intermediate motor units that innervate type IID/X muscle fibres, and (2) activity‐dependent conversion of motor units to those innervated by smaller motor axons innervating type IIA fatigue‐resistant muscle fibres.

Rolipram-induced elevation of cAMP or chondroitinase ABC breakdown of inhibitory proteoglycans in the extracellular matrix promotes peripheral nerve regeneration.

The inhibitory growth environment of myelin and extracellular matrix proteoglycans in the central nervous system may be overcome by elevating neuronal cAMP or degrading inhibitory proteoglycans with chondroitinase ABC (ChABC). In this study, we asked whether similar mechanisms operate in peripheral nerve regeneration where effective Wallerian degeneration removes myelin and extracellular proteoglycans slowly. We repaired transected common peroneal (CP) nerve in rats and either elevated cAMP in the axotomized neurons by subcutaneous rolipram, a specific inhibitor of phosphodiesterase IV, and/or promoted degradation of proteoglycans in the distal nerve stump by local ChABC administration. Rolipram treatment significantly increased the number of motoneurons that regenerated axons across the repair site at 1 and 2 weeks, and increased the number of sensory neurons that regenerated axons across the repair site at 2 weeks. Local application of ChABC had a similar effect to rolipram treatment in promoting motor axon regeneration, the effect being no greater when rolipram and ChABC were administered simultaneously. We conclude that blocking inhibitors of axon regeneration by elevating cAMP or degrading proteoglycans in the distal nerve stump promotes peripheral axon regeneration after surgical repair of a transected nerve. It is likely that elevated cAMP is sufficient to encourage axon outgrowth despite the inhibitory growth environment such that simultaneous enzymatic proteoglycan degradation does not promote more axon regeneration than either elevated cAMP or proteoglycan degradation alone.

Effect of exercise on stability of chronically enlarged motor units.

Chronic denervation syndromes such as the post‐polio syndrome are associated with progressive muscle weakness and fatigue after motoneuron death. Neither the etiology nor the management of these syndromes is clear. To address this issue, we partially denervated rat hindlimb muscles for 1 or 12 months and examined whether chronically enlarged motor units (MUs) become destabilized with time and further destabilized by daily running on exercise wheels. MU enlargement, measured electrophysiologically and morphologically was significantly reduced at 12 months in extensively denervated muscles, and to a lesser extent in moderately denervated muscles, as compared to the findings at 1 month. A 1‐month period of running exercise further reduced the size of the chronically enlarged MUs in the extensively denervated muscles. We have therefore (1) successfully established a rat model of time‐related MU size reduction, in which destabilization of chronically enlarged MUs results in loss of axonal terminals, and (2) demonstrated that nonphysiological activity has small but significant effects of further destabilizing the chronically enlarged MUs.

Brief electrical stimulation improves nerve regeneration after delayed repair in Sprague Dawley rats

Functional recovery after peripheral nerve injury and surgical repair declines with time and distance because the injured neurons without target contacts (chronic axotomy) progressively lose their regenerative capacity and chronically denervated Schwann cells (SCs) atrophy and fail to support axon regeneration. Findings that brief low frequency electrical stimulation (ES) accelerates axon outgrowth and muscle reinnervation after immediate nerve surgery in rats and human patients suggest that ES might improve regeneration after delayed nerve repair. To test this hypothesis, common peroneal (CP) neurons were chronically axotomized and/or tibial (TIB) SCs and ankle extensor muscles were chronically denervated by transection and ligation in rats. The CP and TIB nerves were cross-sutured after three months and subjected to either sham or one hour 20Hz ES. Using retrograde tracing, we found that ES significantly increased the numbers of both motor and sensory neurons that regenerated their axons after a three month period of chronic CP axotomy and/or chronic TIB SC denervation. Muscle and motor unit forces recorded to determine the numbers of neurons that reinnervated gastrocnemius muscle demonstrated that ES significantly increased the numbers of motoneurons that reinnervated chronically denervated muscles. We conclude that electrical stimulation of chronically axotomized motor and sensory neurons is effective in accelerating axon outgrowth into chronically denervated nerve stumps and improving target reinnervation after delayed nerve repair. Possible mechanisms for the efficacy of ES in promoting axon regeneration and target reinnervation after delayed nerve repair include the upregulation of neurotrophic factors.

Chemical communication between regenerating motor axons and Schwann cells in the growth pathway

There are receptors on denervated Schwann cells that may respond to the neurotransmitters that are released from growth cones of regenerating motor axons. In order to ascertain whether the interaction of the transmitters and their receptors plays a role during axon regeneration, we investigated whether pharmacological block of the interaction would reduce the number of motoneurons that regenerate their axons after nerve section and surgical repair. Peripheral nerves in the hindlimbs of rats and mice were cut and repaired, and various drugs were applied to the peripheral nerve stump either directly or via mini‐osmotic pumps over a 2–4‐week period to block the binding of acetylcholine to nicotinic and muscarinic acetylcholine receptors (AChRs: α‐bungarotoxin, tubocurarine, atropine and, gallamine) and binding of ATP to P2Y receptors (suramin). In rats, the nicotinic AChR antagonistic drugs and suramin reduced the number of motoneurons that regenerated their axons through the distal nerve stump. In mice, suramin significantly reduced the upregulation of the carbohydrate HNK‐1 on the Schwann cells in the distal nerve stump that normally occurs during motor axon regeneration. These data indicate that chemical communication between regenerating axons and Schwann cells during axon regeneration via released neurotransmitters and their receptors may play an important role in axon regeneration.

Side-to-side nerve grafts sustain chronically denervated peripheral nerve pathways during axon regeneration and result in improved functional reinnervation.

BACKGROUND:Progressive atrophy of Schwann cells in denervated nerve stumps is a major reason for progressive failure of functional recovery after peripheral nerve injury and surgical repair. OBJECTIVE:To examine whether side-to-side nerve bridges between an intact donor nerve and a recipient denervated distal nerve stump promote nerve growth and in turn, protect distal nerve stumps to improve axon regeneration after delayed surgical repair. METHODS:In Sprague-Dawley rats, 1 or 3 side-to-side common peroneal (CP) nerve bridges were used to bridge between the donor intact tibial (TIB) nerve and a recipient denervated CP distal nerve stump in the contralateral hind limb. No bridges were placed in control animals. After 4 months, either a fluorescent retrograde dye was applied to back-label TIB motoneurons with axons that had grown into the CP nerve stump or the proximal and distal CP nerve stumps were resutured in experimental and control animals to encourage CP nerve regeneration for 5 months. Retrograde dyes were again applied to count CP motoneurons that regenerated their axons through protected and unprotected nerve stumps. RESULTS:Significantly more donor TIB motoneurons regenerated axons into the recipient denervated CP nerve stump through 3 side-to-side CP nerve bridges compared with 1 bridge. This TIB nerve protection significantly increased the number of CP motoneurons regenerating axons through the denervated CP nerve stumps, the number of regenerated axons, and the weight of the reinnervated muscles. CONCLUSION:Multiple side-to-side nerve bridges protect chronically denervated nerve stumps to improve axon regeneration and target reinnervation after delayed nerve repair.

Reliability of isolated isometric function measures in rat muscles composed of different fibre types

The present study investigated the absolute reliability (RAb) of isometric measures of time‐to‐peak tension (TTP), half‐rise time (½RT), half‐fall time (½FT), twitch force (TWf) tetanic force (TETf) and the sag ratio as applied to the slow soleus (SOL) and the fast‐twitch extensor digitorum longus (EDL) and medial gastrocnemius (MG) muscles of the rat hindlimb. In addition, the relationship of each individual isometric measure was examined with regard to the pattern of myosin heavy chain (MHC) isoform expression. Measures of TTP, ½RT, ½FT and sag ratio were negatively correlated with MHCIId(x) and MHCIIb (P < 0.0001), and positively correlated with MHCI (P < 0.0001). TWf and TETf were negatively correlated with MHCI content (P < 0.0001) and positively with MHCIId(x) (P < 0.0001) and MHCIIb (P < 0.001). Comparisons of isometric measures using a paired Students t test revealed that they were not different between the right and left legs; all measures displayed high correlations between the left and right legs (r= 0.71–0.85, P < 0.0001). In contrast to standard tests of statistical significance, these functional measures exhibited a considerable range of RAb when individual muscles were studied in only one hindlimb. When averaged across all muscles, however, the ½FT, ½RT, TWf and TTP measures possessed high overall reliability; measures of TETf and sag ratio were moderately reliable. The results of this study show that the isometric measures studied possess significant predictive value with regard to MHC isoform content; the left and right legs are interchangeable but display a considerable range of reliability when only one hindlimb is studied.

Tetrodotoxin prevents motor unit enlargement after partial denervation in rat hindlimb muscles.

Findings that increased neuromuscular activity significantly reduced sprouting in partially denervated muscles prompted this present study to determine if the converse is true, namely that reduced activity promotes sprouting and motor unit (MU) enlargement. Partial denervation of rat hindlimb muscles by either the L4 or L5 spinal root avulsion resulted in extensive denervation (> 80%) in tibialis anterior (TA) and medial gastrocnemius (MG) muscles, and moderate denervation (∼50%) in soleus (SOL) and plantaris (PL) muscles. The partially denervated muscles were then subjected to a 4 week programme of normal caged activity or TTX‐induced neuromuscular inactivity. At 1 month, measurement of MU enlargement and quantification of sprouting were evaluated, respectively, by electrophysiological and histochemical means. Analysis of electrophysiological data showed that MU forces were significantly increased in both extensively and moderately denervated muscles 1 month after partial denervation and normal cage activity and that neuromuscular activity blockade by TTX completely abolished the MU enlargement in these partially denervated muscles. Histochemical analysis of sprouting revealed that the number of sprouts was significantly increased after partial denervation and normal cage activity, particularly after extensive denervation. TTX‐induced neuromuscular inactivity dramatically reduced the number of sprouts and increased the number of free endplates in the extensively but not the moderately denervated muscles. These data demonstrate that a reduction in neuromuscular activity mediated by presynaptic blockade of neural action potentials reduces MU enlargement in partially denervated muscles by reducing axonal sprouting.

Adaptive responses to creatine loading and exercise in fast-twitch rat skeletal muscle

We investigated the effects of chronic creatine loading and voluntary running (Run) on muscle fiber types, proteins that regulate intracellular Ca2+, and the metabolic profile in rat plantaris muscle to ascertain the bases for our previous observations that creatine loading results in a higher proportion of myosin heavy chain (MHC) IIb, without corresponding changes in contractile properties. Forty Sprague-Dawley rats were assigned to one of four groups: creatine-fed sedentary, creatine-fed run-trained, control-fed sedentary, and control-fed run-trained animals. Proportion and cross-sectional area increased 10% and 15% in type IIb fibers and the proportion of type IIa fibers decreased 11% in the creatine-fed run-trained compared with the control-fed run-trained group (P < 0.03). No differences were observed in fast Ca2+-ATPase isoform SERCA1 content (P > 0.49). Creatine feeding alone induced a 41% increase (P < 0.03) in slow Ca2+-ATPase (SERCA2) content, which was further elevated by 33% with running (P < 0.02). Run training alone reduced parvalbumin content by 50% (P < 0.05). By comparison, parvalbumin content was dramatically decreased by 75% (P < 0.01) by creatine feeding alone but was not further reduced by run training. These adaptive changes indicate that elevating the capacity for high-energy phosphate shuttling, through creatine loading, alleviates the need for intracellular Ca2+ buffering by parvalbumin and increases the efficiency of Ca2+ uptake by SERCAs. Citrate synthase and 3-hydroxyacyl-CoA dehydrogenase activities were elevated by run training (P < 0.003) but not by run training + creatine feeding. This indicates that creatine loading during run training supports a faster muscle phenotype that is adequately supported by the existing glycolytic potential, without changes in the capacity for terminal substrate oxidation.