tian Wu

Expression of Nitric-Oxide Synthase (NOS) in Injured CNS Neurons as Shown by NADPH Diaphorase Histochemistry

Recent studies have implicated nitric oxide (NO) in several mechanisms related to neuronal degeneration and synaptic plasticity. In the present study, two models of traumatic neuronal injury were used to examine the expression of NOS following neuronal injury and its relationship to axonal sprouting and neuronal degeneration. It was found that NOS is induced in a week of axonal injury in neurons that are normally NOS-negative. Spinal motoneurons express the enzyme after ventral root avulsion, but not after ventral root transection. Neurons of the nucleus dorsalis of the spinal cord express NOS after ipsilateral spinal cord hemisection. These two models provide information about the time course of NOS expression in injured neurons and the opportunity in future studies to determine the role of NOS and its product, NO, in CNS injury. Observations from the present study suggest that early NOS expression seems to be associated with axonal sprouting and growth. Interestingly, though, the neurons expressing lesion-induced NOS ultimately die. Whether NOS expression in these cells is related to their death is currently under investigation.

Neuronal nitric oxide synthase is induced in spinal neurons by traumatic injury.

Nitric oxide appears to mediate the immune functions of macrophages, the influence of endothelial cells on blood vessel relaxation, and also to serve as a neurotransmitter in the central and peripheral nervous system. Macrophage nitric oxide synthase is inducible with massive increases in new nitric oxide synthase protein synthesis following immune stimulation of macrophages. By contrast, endothelial nitric oxide synthase and neuronal nitric oxide synthase are thought to be constitutive with activation induced by calcium entry into cells in the absence of new protein synthesis. Developmental studies showing the transient expression of neuronal nitric oxide synthase in embryonic and early postnatal life in rodent spinal motoneurons and cerebral cortical plate neurons (Bredt and Snyder, unpublished observations) implies inducibility of neuronal nitric oxide synthase. Moreover, neuronal nitric oxide synthase expression is greatly enhanced in sensory ganglia following peripheral axotomy. Staining for NADPH diaphorase in spinal motoneurons is greatly increased following ventral root avulsion. In many parts of the Central Nervous System NADPH diaphorase staining reflects nitric oxide synthase. In the present study, we have combined in situ hybridization for neuronal nitric oxide synthase, immunohistochemical staining of neuronal nitric oxide synthase, and NADPH diaphorase staining to establish that neuronal nitric oxide synthase expression is markedly augmented in spinal motoneurons following avulsion. The generality of this effect is evident from augmented staining in nucleus dorsalis following spinal cord transection.

Potential Roles of Gene Expression Change in Adult Rat Spinal Motoneurons Following Axonal Injury: A Comparison among c-jun, Low-Affinity Nerve Growth Factor Receptor (LNGFR), and Nitric Oxide Synthase (NOS)

The present study investigates expression of nitric oxide synthase (NOS), immediate early genes (IEGs, c-jun, and c-fos), and low-affinity nerve growth factor receptor (LNGFR) in adult rat spinal motoneurons in response to three conditions of axonal injury: distal axotomy, root avulsion, and root avulsion followed by a peripheral nerve (PN) graft implantation. Expression of c-jun and LNGFR were predominately observed in motoneurons of the distal axotomized segment where most motoneurons survived. In contrast, expression of NOS was exclusively found in motoneurons of the rootavulsed segment where most motoneurons died. c-fos was not expressed in motoneurons following either distal axotomy or root avulsion. In animals with PN graft implantation, a double fluorescent labeling technique was used to evaluate motoneuron regeneration. Expression of NOS was completely inhibited in all motoneurons that regenerated into the PN graft, but was not inhibited in those that did not regenerate. Moreover, regenerated motoneurons expressed LNGFR and c-jun while the nonregenerated motoneurons expressed NOS. Results of the present study have shown that motoneurons undergo changes in expression of cellular molecules in response to the axonal injury. The expression of c-jun and LNGFR may be related to the regenerative process while expression of NOS is more likely involved in the degenerative process. The results also show that PN graft implantation can alter the expression of cellular molecules and reduce motoneuron death due to root avulsion. The survival-promoting effects of PN graft implantation (presumably the effects of neurotrophic factors) may be achieved by modifying certain cellular molecules such as NOS.

Implantation of PNS graft inhibits the induction of neuronal nitric oxide synthase and enhances the survival of spinal motoneurons following root avulsion

In a spinal root injury model, our previous studies have shown that induction of nitric oxide synthase (NOS) appears only in spinal motoneurons of the root-avulsed segment in which significant motoneuron loss occurs but not in those of the distal root-axotomized segment (root axotomy 5-10 mm from the spinal cord) in which most motoneurons survive the injury. One hypothesis for the different response of motoneurons to root avulsion and distal root axotomy is that neurotrophic factors produced by the remaining peripheral nervous system (PNS) component are available for the distally axotomized motoneurons but are not available following avulsion. This hypothesis is tested in the present study by implantation of a PNS graft following the root avulsion. Results of the present study show that implantation of a PNS graft significantly enhances the survival of motoneurons following avulsion. Expression of NOS due to avulsion injury is completely inhibited in all motoneurons that regrow into the PNS graft. These results indicate that induction of NOS in avulsed motoneurons may result from the deprivation of neurotrophic factors produced by the PNS component, and the survival promoting effects of neurotrophic factors may be achieved by modifying certain cellular molecules such as NOS.

Expression of c-jun and neuronal nitric oxide synthase in rat spinal motoneurons following axonal injury

Expression of neuronal NOS, c-jun and c-fos in spinal motoneurons following axonal damage were investigated in the present study. Although either distal spinal root axotomy or root avulsion induced expression of c-jun, expression of c-jun was predominantly found in distal root-axotomized motoneurons. In contrast, expression of NOS was exclusively observed in avulsed motoneurons. c-fos was not expressed in spinal motoneurons following either distal root axotomy or root avulsion. The different expression patterns of c-jun and NOS in the injured neurons suggest that these molecules may involve in different cellular processes and might play different roles in response to the injury. Since distal root axotomy did not cause motoneuron death and root avulsion did, expression of c-jun is likely related to regenerative process while induction of NOS may be involved in the degenerative process.

Induction of nitric oxide synthase and motoneuron death in newborn and early postnatal rats following spinal root avulsion

It is well established that immature motoneurons are more vulnerable to axonal injury than the adult ones. Many previous studies used distal axonal injury, such as sciatic nerve transection, to examine the response of immature motoneurons to injury. In the present study, a more severe injury, spinal root avulsion, was performed in newborn and early postnatal rats, and the response of immature spinal motoneurons to the injury was observed. In newborn rats, root avulsion causes motoneuron loss which is similar to that of previous studies by distal axonal injury. During the early postnatal development, motoneuron loss in immature rats is greater following root avulsion when compared to cell loss following distal axonal injury at any given age. Following root avulsion, nitric oxide synthase (NOS) is induced in injured immature motoneurons. The induction and accumulation of NOS in injured motoneurons is much more rapid in the immature animals than in the adult, which is coincident with the more rapid motoneuron loss in the immature animals. Results of the present study indicate an involvement of NOS in neuronal degeneration. However, the precise role of NOS in the response of motoneurons to axonal injury is not clear and needs to be further studied.

Increased Expression of Nitric Oxide Synthase in Hypothalamic Neuronal Regeneration

This investigation deals with the histochemical and scanning electron microscopic (SEM) correlates that depict regeneration of the neurohypophyseal system that may be nitric oxide dependent following hypophysectomy in the rodent hypothalamus. NOS histochemistry and correlative SEM were employed to establish the rates of regrowth and appearance of NOS-positive supraoptic (SON) and paraventricular (PVN) neurites and their cell bodies following hypophysectomy. NOS activity increased significantly in SON and PVN neuronal perikarya and regenerating axons by 2 weeks. NOS-positive neurites were observed to regrow into the adjacent median eminence and insinuate into the lumen of the third cerebral ventricle. By 4 weeks posthypophysectomy, NOS staining of SON and PVN neurons and their regrown neurites had returned to normal control levels. Despite this fact, large complexes of apparent magnocellular neurites remained upon the floor of the third cerebral ventricle as observed with SEM. These observations support the hypothesis that NO may play a fundamental role in the process of regeneration, plasticity, and retargeting of SON and PVN axons following injury.

Development of nitric oxide synthase expression in the superficial dorsal horn of the rat spinal cord

Development of nitric oxide synthase (NOS) expression in the superficial dorsal horn of the rat spinal cord was studied using NADPH diaphorase histochemistry. At birth, no positive staining was seen in the superficial laminae of the cord. A week later, a few small positive neurons and fibers were seen in presumptive lamina II. The adult pattern of NOS expression was evident by the end of the third postnatal week.

Neural regeneration and neuronal migration following injury. I. The endocrine hypothalamus and neurohypophyseal system

Central to this investigation are several basic hypotheses that are designed to test the role of nitric oxide (NO) in the complex process of central regeneration and plasticity in a well established model system of the mammalian brain. We have employed histochemical techniques at the light and ultrastructural level coupled with correlative scanning electron microscopy, immunoelectron microscopy, and in situ hybridization in order to determine the functional significance of the increased expression of nitric oxide synthase (NOS) in neurons of the supraoptic (SON) and paraventricular (PVN) nuclei which accompanies regeneration of their axotomized neurites following hypophysectomy. The aim of this investigation was to determine the potential role and temporal up-regulation of NOS in this basic regenerative process and to establish the ultrastructural and neuroanatomical correlates during critical periods of regeneration and regrowth of SON and PVN axons following hypophysectomy in the endocrine hypothalamus of the rat. Our data support the hypothesis that NO may serve as a second messenger molecule that may act in some fashion to govern not only the process of central regeneration and regrowth of magnocellular (SON/PVN) axons into the median eminence, neural stem, and neural lobe (the neurohypophyseal system) but may also influence the regeneration of neurites into new neuroanatomical domains such as the adjacent lumen of the third cerebral ventricle. We have demonstrated a distinct temporal relationship between injury (axotomy) of SON/PVN axons and the establishment of new neurovascular zones following hypophysectomy with the up-regulation of NOS. This up-regulation appears to correlate well with successful regeneration in the mammalian neurohypophyseal system. We have also successfully inhibited axonal regeneration with the use of nitroarginine, a competitive antagonist of NO. NOS up-regulation attendant to regeneration of SON and PVN axons may have inestimable clinical implications, particularly with respect to closed head injury and cerebral contusion that involves the mechanical shearing of the infundibular stalk. In addition, this investigation has reaffirmed that large numbers of bona fide neurons migrate and emerge upon the floor of the adjacent third cerebral ventricle shortly following hypophysectomy (within 2 weeks). The origin and mechanisms of neuronal migration and plasticity following hypophysectomy are the subject of interpretation and discussion in this investigation.

Transplantation of the Mammalian Pineal Gland: Studies of Survival, Revascularization, Reinnervation, and Recovery of Function

The survival, revascularization, reinnervation, and recovery of function of transplanted rat pineal glands were studied following grafting into four different locations in pinealectomized rats. Pineal grafts were well vascularized by fenestrated capillaries. Pinealocytes in the grafts maintained high-metabolic activity. More nerve fibers and terminals were observed in the grafts within the anterior chamber of the eye than in the third cerebral ventricle and the pineal region (in situ transplantation). No fibers or terminals were found in grafts placed beneath the renal capsule. Nighttime serum melatonin levels increased significantly in pinealectomized rats with transplants into either the third cerebral ventricle or the anterior eye chamber. This increase might reflect graft reinnervation. Yet day-night differences in serum melatonin were observed only in host rats receiving transplants in the anterior eye chamber. In conclusion, pinealocytes survived transplantation into different locations and exhibited ultrastructural features indicative of active secretory processes; however, day-night differences in serum melatonin are only restored following transplants into the anterior eye chamber. Reinnervation of the grafts by the host superior cervical ganglion is necessary for this restoration.