Synergistic motor nerve fiber transfer between different nerves through the use of end-to-side coaptation.

End-to-end nerve repair is a widely used and successful experimental microsurgical technique via which a denervated nerve stump is supplied with reinnervating motor or sensory axons. On the other hand, questions are still raised as concerns the reliability and usefulness of the end-to-side coaptation technique. This study had the aim of the reinnervation of the denervated forearm flexor muscles in baboons through the use of an end-to-side coaptation technique and the synergistic action of the radial nerve. The median and ulnar nerves were transected, and the motor branch of the radial nerve supplying the extensor carpi radialis muscles (MBECR) was used as an axon donor for the denervated superficial forearm flexors. A nerve graft was connected to the axon donor nerve through end-to-side coaptation, while at the other end of the graft an end-to-end connection was established so as to reinnervate the motor branch of the forearm flexors. Electrophysiological investigations and functional tests indicated successful reinnervation of the forearm flexors and recovery of the flexor function. The axon counts in the nerve segments proximal (1038+/-172 S.E.M.) and distal (1050+/-116 S.E.M.) to the end-to-side coaptation site and in the nerve graft revealed that motor axon collaterals were given to the graft without the loss or appreciable misdirection of the axons in the MBECR nerve distal to the coaptation site. The nerve graft was found to contain varying, but satisfactory numbers of axons (269+/-59 S.E.M.) which induced morphological reinnervation of the end-plates in the flexor muscles. Accordingly, we have provided evidence that end-to-side coaptation can be a useful technique when no free donor nerve is available. This technique is able to induce limited, but still useful reinnervation for the flexor muscles, thereby producing a synergistic action of the flexor and extensor muscles which allows the hand to achieve a basic gripping function.

Delayed riluzole treatment is able to rescue injured rat spinal motoneurons.

The effect of delayed 2-amino-6-trifluoromethoxy-benzothiazole (riluzole) treatment on injured motoneurons was studied. The L4 ventral root of adult rats was avulsed and reimplanted into the spinal cord. Immediately after the operation or with a delay of 5, 10, 14 or 16 days animals were treated with riluzole (n=5 in each group) while another four animals remained untreated. Three months after the operation the fluorescent dye Fast Blue was applied to the proximal end of the cut ventral ramus of the L4 spinal nerve to retrogradely label reinnervating neurons. Three days later the spinal cords were processed for counting the retrogradely labeled cells and choline acetyltransferase immunohistochemistry was performed to reveal the cholinergic cells in the spinal cords. In untreated animals there were 20.4+/-1.6 (+/-S.E.M.) retrogradely labeled neurons while in animals treated with riluzole immediately or 5 and 10 days after ventral root avulsion the number of labeled motoneurons ranged between 763+/-36 and 815+/-50 (S.E.M.). Riluzole treatment starting at 14 and 16 days after injury resulted in significantly lower number of reinnervating motoneurons (67+/-4 and 52+/-3 S.E.M., respectively). Thus, riluzole dramatically enhanced the survival and reinnervating capacity of injured motoneurons not only when treatment started immediately after injury but also in cases when riluzole treatment was delayed for up to 10 days. These results suggest that motoneurons destined to die after ventral root avulsion are programmed to survive for some time after injury and riluzole is able to rescue them during this period of time.

Ischemia increases prostaglandin H synthase-2 levels in retina and visual cortex in piglets.

BACKGROUND: Ischemia increases levels of prostaglandin H synthase-2 (PGHS-2) in neonatal brain and cerebral vasculature, but effects on the developing visual system are unknown. We examined the effects of ischemia on PGHS-2 mRNA and protein levels in the retina and visual cortex in anesthetized piglets. METHODS: Ten minutes of complete retinal and brain ischemia was induced by increasing intracranial pressure. After 2-12 h of reperfusion, samples of retina and visual cortex were collected for determinations of levels of PGHS-2 mRNA (RNase protection assay) or protein (immunohistochemistry and western blotting). Tissues also were obtained from control animals. RESULTS: Levels of PGHS-2 mRNA were undetectable in control animals but showed a dramatic increase at 2-4 h in the cortex and retina in animals exposed to ischemia. Detectable but limited PGHS-2 immunoreactivity (IR) was present in the retina and visual cortex from control animals. In piglets not subjected to ischemia, PGHS-2 IR was localized mainly to the outer limiting membrane and to the Muller cells. Ischemia induced a marked increase in PGHS-2 IR in the neural retina, with the greatest increase in the photoreceptor layer. PGHS-2 levels in whole retina also increased at 8 h after ischemia. In the intact visual cortex PGHS-2 IR was evident in layers II and V. Ischemia increased the intensity of IR in layers II/III as well as layer V. CONCLUSIONS: Detectable amounts of PGHS-2 protein are present in the piglet retina and visual cortex under normal conditions, but levels are markedly increased 8-12 h after ischemic stress. Enhanced PGHS-2 levels after ischemic stress may contribute to delayed pathological changes of the visual system in the neonate.

The effect of riluzole treatment in rats on the survival of injured adult and grafted embryonic motoneurons.

The effect of riluzole on the survival of injured motoneurons was studied. The L4 ventral root was avulsed and reimplanted into the spinal cord. Immediately after the operation, 4 animals were treated with riluzole for 3 weeks while another 4 animals received no treatment after the operation. Three months later the fluorescent dyes, Fast Blue and Diamidino Yellow, were applied to the cut ventral ramus of the L4 spinal nerve, for retrograde labelling of neurons. Three days later, the spinal cords were processed to reveal the retrograde-labelled cells. In untreated animals, there were 20 +/- 2.1 labelled neurons (+/- SEM), while in animals treated with riluzole there were 723 +/- 26. Thus, treatment with riluzole dramatically enhanced the survival of injured motoneurons. In another series of experiments, after avulsion of the L4 ventral root and its reinsertion, embryonic spinal cord pieces were grafted into the host cord. Five animals received riluzole treatment and 4 were left untreated. In the untreated animals, 125 +/- 5.1 retrograde-labelled cells of both graft and host origin were detected. In rats treated with riluzole, 645 +/- 35.7 retrograde-labelled cells were seen and almost all of these were of host origin. Thus, treatment with riluzole enhanced the survival of injured host motoneurons, and by doing so, (i) reduced the ability of grafted neurons to extend their axons into the reimplanted L4 ventral root, and (ii) reduced the survival of the grafted cells.

Atomic force microscopic study of the human cornea following excimer laser keratectomy.

The aim of this study was to examine the corneal surface structures with a new investigative method, the atomic force microscope following 193 nm excimer laser photoablation. Fresh human corneas were irradiated in vitro with an increasing number of impulses emitted by a 193 nm ArF laboratory excimer laser in order to produce either smooth flat surfaces or stair-like formations within the cornea. The corneas were investigated in a Topometrix(R) atomic force microscope in their native state. For comparison, three corneas were fixed with glutaraldehyde and processed for scanning electron microscopy. Atomic force microscopy and scanning electron microscopy revealed the same surface characteristics of photoablated corneas, though the preparation for scanning electron microscopy induced considerable shrinkage of the tissues. The layers of the cornea could be distinguished from each other and deeper ablations of the stroma produced a rougher surface. On the lateral walls of ablated stairs small droplets of ejected material could be seen with scanning electron microscope. Atomic force microscope produces three-dimensional images of the scanned native corneal surfaces and it could be a valuable tool to investigate the corneal smoothness. Our investigations have provided similar results as those obtained with scanning electron microscopy showing that the laser-ablated corneal surface remains relatively smooth. We suggest that the formation of condense droplets of ejected materials is based on hydrodynamic motions induced by boiling water solutions.

Carbonic anhydrase II and carbonic anhydrase-related protein in the cerebellar cortex of normal and lurcher mice.

The developmental profiles of carbonic anhydrase II (CA-II) and a carbonic anhydrase related protein (CARP) were studied in rat and mouse cerebella. Enzyme histochemistry, immunohistochemistry, in situ hybridisation and Western blotting were used to study the synthesis and expression of these enzymes in cerebellar sections from age matched control, CA-II deficient and lurcher mice, the latter being characterised by Purkinje cell degeneration. Both CA-II and CARP were first found to be expressed in the Purkinje cells in the 9 day old mouse, and the immunoreactivity of both peptides increased with time. Immunohistochemistry showed more intense staining of CARP than of CA-II in Purkinje cells throughout the developmental profile of the mouse, and this was mirrored by the mRNA levels determined by in situ hybridisation. Immunohistochemistry of CA-II and CARP also demonstrated the progressive dendritic growth of the mouse and rat Purkinje cells. CA-II and CARP immunoreactivity ceased by the end of cerebellar maturation. The onset of Purkinje cell degeneration was detected at day 10 in the lurcher mouse, with concomitant marked decrease in CA-II level: however CARP expression was found to be unchanged. By postnatal day 16 neither CA-II mRNA, protein, nor activity was detectable in contrast to CARP which remained at a decreased level unit the Purkinje cells population had completely degenerated. Our findings suggest a role of CA-II in the degenerative processes of the lurcher Purkinje cells, with CARP playing an important role in the development and maturation of the cerebellar cortex.

Reinnervation of rat muscles via volkensin-affected and normal peripheral nerve conduits.

Volkensin is a neurotoxic lectin which, when injected into a peripheral nerve is retrogradely transported to the cell body and causes it to die. Accordingly, volkensin-affected peripheral nerves rapidly degenerate. It is, however, not clear whether axonal growth can take place within these degenerated nerves. In this study the ability of volkensin-treated and untreated degenerated peripheral nerves to support regeneration of healthy axons was compared. Four groups of animals were used, in Group 1 the peroneal nerve was cut and 10 days later the proximal stump of the deep tibial nerve was sutured to the distal stump of the peroneal nerve (10 days after axotomy). In the second group of animals the peroneal nerve was treated with volkensin, 10 days later the proximal stump of the deep tibial nerve was connected to the distal section of the cut, thus giving a volkensin-treated peroneal nerve. In the third group, 10 days after the peroneal nerve was treated with volkensin, the proximal stump of the deep tibial nerve was connected directly to the extensor digitorum longus (EDL) muscle. In Group 4 the volkensin-treated peroneal nerve was left intact. Six weeks after surgical intervention the tension of both EDL muscles was recorded and the muscles were processed for histological visualisation of endplates and axons. EDL muscles from Group 1 animals produced 36.5 ± 11.3% S.E.M of maximal tetanic tension produced by the contralateral EDL muscle. Significantly less recovery of function was achieved by EDL muscles in Group 2 animals (9.3 ± 2.5%). Muscles from Group 3, where the healthy nerve was sutured directly into the EDL muscle that had been denervated by volkensin treatment had a significantly better recovery than Group 2 muscles (23 ± 3%). Sprouting of nerve fibres and proliferation of Schwann cells was observed in the muscles from Groups 1 and 3, but not in Group 2. Thus, volkensin-treated peripheral nerves provide a poor conduit for regenerating nerve fibres though muscles denervated, by treatment with volkensin, and can accept reinnervation by healthy nerves. The possible mechanisms that render the volkensin treated peripheral stump a poor conduit for healthy axons is discussed.

Carbonic anhydrase and cardiac pH regulation.

Membrane-bound carbonic anhydrase (CA) has recently been identified in mammalian cardiac tissue. In this study, we have investigated the histochemical location and functional role of CA in the ferret heart. Heart sections stained by a modified Hansson's technique showed CA to be located on capillary endothelial membranes as well as on sarcolemmal membranes. In the Langendorff-perfused heart, washout of CO2 brought about by switching perfusion between 25 mM HCO3(-)-5% CO2-buffered solution and nominally HCO3(-)-CO2-free solution caused a transient rise in intracellular pH (pHi) measured by the chemical shift of 2-deoxy-D-glucose 6-phosphate with 31P nuclear magnetic resonance spectroscopy. The initial rate of change of pHi, measured over the first 60-75 s of CO2 efflux, was significantly reduced from 0.41 +/- 0.03 pH units/min (n = 9) in control hearts to 0.28 +/- 0.02 pH units/min (n = 5) in the presence of the membrane-permeable CA inhibitor 6-ethoxzolamide (P < 0.05 compared with control) and to 0.22 +/- 0.04 pH units/min (n = 5) in the presence of the membrane-impermeable CA inhibitor CL-11,366 (P < 0.01 compared with control). After reperfusion of the ischemic myocardium, both CA inhibitors caused a significant slowing of initial rate of change in pH (and initial rate of recovery of contractile function) compared with control hearts. These results suggest that CA, by facilitating the hydration-dehydration of CO2-H2CO3, alters the relative concentrations of CO2 inside and outside the cells, thus enhancing the rate of CO2 transfer from the intracellular to extracellular compartments, which contributes significantly to pHi recovery after reperfusion of the ischemic myocardium.

Improved motor function of denervated rat hindlimb muscles induced by embryonic spinal cord grafts.

Loss of motoneurons results in a decrease in force production by skeletal muscles and paralysis. Although it has been shown that missing motoneurons of rats can be replaced by embryonic homotopic neurons, attempts to guide their axons to their target muscles that have lost their innervation have been unsuccessful. In this study attempts were made to guide axons from grafted embryonic motoneurons to their target via a reimplanted ventral root. Adult hosts that received an embryonic graft prelabelled with 5-bromo-2'-deoxyuridine had their L4 ventral root avulsed and reimplanted into the spinal cord. Three to six months later, neurons that had their axons in the L4 ventral ramus were retrogradely labelled with fast blue and diamidino yellow. In five animals that had received an embryonic graft 116 +/- 16 cells were retrogradely labelled, and of these at least 15% were of graft origin, since they were positive for 5-bromo-2'-deoxyuridine. In five animals that had their L4 ventral root reimplanted but did not receive a graft, only 12 +/- 1.3 cells were retrogradely labelled. However, meaningful functional recovery could be achieved only if the regenerating axons of embryonic motoneurons found in the L4 ventral ramus were able to reverse the loss of force of muscles that had lost their innervation. This study shows that axons of embryonic motoneurons grafted into an adult rat spinal cord, as well as some axons of host origin, can be guided to denervated hindlimb muscles via reimplanted lumbar ventral roots. In normal rats approximately 30 motor axons innervated the extensor digitorum longus and 60 innervated the tibialis anterior via the L4 ventral root. In rats that did not receive a graft only 3.7 +/- 1.2 axons reached the extensor digitorum longus and 3.5 +/- 0.4 reached the tibialis anterior muscle via the implanted L4 ventral root. In animals that had an embryonic graft, 7.6 +/- 0.5 axons innervated the extensor digitorum longus and 8.5 +/- 0.5 reached the tibialis anterior muscle via the implanted root. In rats without a transplant the maximum tetanic tension elicited by stimulating the implanted L4 root was 16 +/- 7 g for the extensor digitorum longus and 53 +/- 36 g for the tibialis anterior muscle, whereas the corresponding muscles in animals that had an embryonic graft developed 82 +/- 16 and 281 +/- 95 g respectively. Thus it appears that the grafted motoneurons contributed to the innervation and functional recovery of the denervated muscles.

The use of embryonic spinal cord grafts to replace identified motoneuron pools depleted by a neurotoxic lectin, volkensin.

The viability of solid pieces of embryonic spinal cord grafted into adult spinal cords that had been depleted of motoneurons by a neurotoxic lectin, volkensin, was investigated. The possibility that the grafted neurons will extend their axons via the degenerated ventral roots and reinnervate the denervated muscles was also studied. Cells in the embryonic spinal cord were prelabeled with bromodeoxyuridine and grafted into the host cord 2 or 3 weeks after volkensin treatment. Six to 24 weeks later the fluorescent dyes Fast Blue and Diamidino Yellow were applied to the peripheral nerve or muscles which were expected to be reinnervated by neurons from the graft. The grafted cells were visualized by bromodeoxyuridine immunocytochemistry. Although the transplants survived, no neurons of graft origin reinnervated the muscles via the ventral roots of the host spinal cord. Therefore, in another series of experiments axons from grafted and host neurons were guided toward an implanted extensor hallucis longus (EHL) muscle via its motor nerve. The EHL muscle was removed from either healthy or volkensin-affected hindlimb of the host, placed paravertebrally, and the proximal end of its motor nerve inserted into the spinal cord at the site of the graft. In these experiments, neurons of graft origin reinnervated the paravertebrally placed muscle implant via the muscle's own nerve. However, significantly fewer neurons were observed when volkensin-affected nerve-muscle implants were used. To establish whether grafted spinal cord neurons are necessary for reinnervation of skeletal muscles, in separate experiments embryonic neocortical tissue was transplanted into the motoneuron-depleted spinal cord. These grafts were connected to healthy nerve-muscle implants. In this case little reinnervation of the muscle occurred. These results suggest that, though volkensin-affected peripheral nerve is not a good conduit for regenerating axons, other inhibitory effects located in the CNS may be responsible for the failure of growing embryonic neurons to reach their peripheral targets via the existing anatomical routes. Experiments with grafted neocortical tissue indicate that good reinnervation of implanted muscles can only be achieved by using homologous embryonic tissue.

Membrane-bound carbonic anhydrase in human retinal pigment epithelium.

PURPOSE: Inhibition of carbonic anhydrase (CA) by acetazolamide causes a decrease in the standing potential of the retinal pigment epithelium (RPE) and an increase in the rate of subretinal fluid absorption, and it may improve cystoid macular edema. These effects are thought to be mediated by the RPE. Given the solubility coefficient of acetazolamide, the drug is most likely to act by direct inhibition of membrane-bound CA (CA IV). To identify a substrate for acetazolamide in the RPE, the distribution of CA activity and the isoform of CA in the RPE membrane were investigated. METHODS: Carbonic anhydrase activity was determined by Hansson's technique in fresh human eyes from donors of both sexes and different ages. The presence of the membrane-bound isoform CA IV was investigated immunohistochemically at the light and electron microscopic level, as well as by Western blotting in fresh RPE, and in adult and fetal RPE cultures. RESULTS: Hansson's histochemical method demonstrated CA activity on the apical and basolateral cell membrane of the RPE. Using the gamma-globulin fraction of a polyclonal antibody against pure CA IV, immunocytochemistry showed labeling for CA IV on the apical RPE membrane or morphologically polarized human adult and fetal RPE cultures. Gel electrophoresis and Western blotting demonstrated a major immunoreactive band at 55 kDa in homogenates, which was consistently reduced to approximately 35 kDa by incorporation of 0.1% Triton X-100 detergent. CONCLUSIONS: These results suggest that the clinical effects of carbonic anhydrase inhibitors on RPE function may be mediated via membrane-bound carbonic anhydrase activity in RPE and that CA IV is responsible for activity on the apical surface.

Pharmacological inhibition of brain carbonic anhydrase protects against 4-aminopyridine seizures.

The effects of the inhibition of carbonic anhydrase on the manifestation of tonic-clonic seizures were investigated in freely-moving rats. 4-Aminopyridine, a specific blocker of the neuronal K+ channels was used to produce generalized convulsions. After pretreatment of adult male rats with 20 or 40 mg/kg acetazolamide, 3, 5, 7 or 9 mg/kg 4-aminopyridine was injected intraperitoneally and the latencies of the symptoms were measured. Pharmacological inhibition of brain carbonic anhydrase significantly increased the latency of onset of seizures. Bolus administration of acetazolamide decreased the incidence of generalized convulsions and protected against status epilepticus. Chronic acetazolamide treatment for 2 days affected only the generalized convulsions. The results suggested that alterations of the extracellular and intracellular pH by acetazolamide decreased the efficacy of synaptic transmission in several areas of the brain. The possible effects of the HCO3- ions on the sensitivity of synaptic and nonsynaptic neuronal receptors are discussed.

Differential expression of carbonic anhydrase isozymes in microglial cell types.

Microglia cells have been shown to express carbonic anhydrase. Using carbonic anhydrase histochemistry and immunohistochemistry, different types of central nervous system microglial cells were detected, which expressed two main carbonic anhydrase (CA) isozymes during the early postnatal stage of development and after peripheral nerve injury in the spinal cord of adult rats. Amoeboid and reactive microglial cells were heavily immunostained for CA-II and CA-III and showed colocalization with complement receptor type 3 and Griffonia Simplicifolia B4 isolectin. Resting microglial cells in the brain and spinal cord showed faint CA-III staining and were negative for CA-II. These results show that not only CA-II, but also CA-III isozyme is represented in the central nervous system and carbonic anhydrase activity may correlate with metabolic and immunological changes of microglial cells. These data also further strengthen the idea of the mesodermal origin of central nervous system macrophages.

Localization of acetazolamide-resistant carbonic anhydrase III in human and rat choroid plexus by immunocytochemistry and in situ hybridisation.

Carbonic anhydrase is an essential metabolic enzyme of the central nervous system and has an important role in the production and regulation of cerebrospinal fluid. Although it has been known for over 30 years that inhibition of the enzyme with acetazolamide dramatically but not completely reduces the production of cerebrospinal fluid, the precise mechanism of the inhibitory action has been only recently revealed. In this study we present evidence that apart from carbonic anhydrase II, the catalytically highly active isozyme, carbonic anhydrase III, an acetazolamide-resistant and kinetically different isozyme could be demonstrated in the epithelial cells of the developing and mature rodent and human choroid plexuses. Both isozymes express intense immunostaining revealed with specific antisera, and by using in situ hybridisation histochemistry, carbonic anhydrase III mRNA was also observed. Since the kinetic properties and proportion of brain carbonic anhydrase III in the human choroid plexus are not revealed the function of this isozyme in choroid plexus is still to be determined.

The use of a neurotoxic lectin, volkensin, to induce loss of identified motoneuron pools.

In this study we investigated degeneration of defined motor pools in the adult rat spinal cord and the associated changes in spinal cord in dorsal root ganglia and peripheral nerve. Degeneration of motoneurons was induced by the neurotoxic lectin, volkensin. This substance is taken up by the axons and retrogradely transported to the cell body, where it inhibits proteosynthesis and kills the neuron. Accordingly, in adult Wistar rats the peroneal or the sciatic nerve was injected with 5.0 ng volkensin, and the effect of this single injection was investigated at different intervals after the operation. Retrograde labelling by horseradish peroxidase was used to reveal the extent of cell death and glial repair was studied by immunostaining with different glial cell markers. Degenerating cells were observed in the ventral horn of the lumbar spinal cord and L4 and L5 dorsal root ganglia as early as four days after volkensin treatment and by two weeks no retrogradely labelled motoneurons could be found in the treated peroneal pool. These changes were accompanied by severe muscle weight loss. Examination of the ventral horn of the spinal cord on the treated side revealed many hypertrophic astrocytes and reactive microglial cells expressing an increased level of complement receptor type 3 immunoreactivity. In the volkensin-injected peripheral nerve, distinct signs of Wallerian-like degeneration could be observed. Schwann cells identified by immunostaining to S-100 protein appeared to be preserved. Interestingly, at later stages after volkensin injection (four to eight weeks), some retrogradely labelled motoneurons were seen in the peroneal pool; their number occasionally reached 18.4% of the control pool. The dorsal root ganglia showed extensive loss of neurons and numerous abnormal neurons were found throughout the period of the study. These findings suggest that some motoneurons are able to recover from exposure to volkensin and temporary arrest of proteosynthesis. Despite this, volkensin-induced selective motoneuron death in the adult rat can be a useful experimental model for degenerative motoneuron disease.

Nucleotide sequence and chromosomal assignment of a cDNA encoding the large isoform of human glutamate decarboxylase.

Glutamic acid decarboxylase (GAD) catalyses the conversion of L-glutamic acid to the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). Two forms of human GAD, GAD65 and GAD67, are encoded by two separate genes. A full length human GAD67 cDNA has been isolated from a human frontal cortex cDNA library and the nucleotide sequence determined. The GAD67 gene has been mapped to chromosome 2 using the polymerase chain reaction to amplify specifically the human sequence in rodent/human somatic cell hybrid DNA. This confirms that human GAD67 is not syntenic with the smaller GAD isoform GAD65 which has been assigned to chromosome 10. Production of polyclonal antiserum to a baculovirus-expressed GAD67 enabled immunocytological detection of GAD in the rat brain.