Robust axonal growth and a blunted macrophage response are associated with impaired functional recovery after spinal cord injury in the MRL/MpJ mouse.

Spinal cord injury (SCI) in mammals leads to a robust inflammatory response followed by the formation of a glial and connective tissue scar that comprises a barrier to axonal regeneration. The inbred MRL/MpJ mouse strain exhibits reduced inflammation after peripheral injury and shows true regeneration without tissue scar formation following an ear punch wound. We hypothesized that following SCI, the unique genetic wound healing traits of this strain would result in reduced glial and connective tissue scar formation, increased axonal growth, and improved functional recovery. Adult MRL/MpJ and C57BL/6J mice were subjected to a mid-thoracic spinal contusion and the distribution of axon profiles and selected cellular and extracellular matrix components was compared at 1, 2, 4 and 6 weeks post-injury. Recovery of hind-limb locomotor function was assessed over the same time period. The MRL/MpJ mice exhibited robust axon growth within the lesion, beginning at 4 weeks post-injury. This growth was accompanied by reduced macrophage staining at 1, 2, 4 and 6 weeks post-injury, decreased chondroitin sulfate proteoglycan staining at 1-2 weeks and increased laminin staining throughout the lesion at 2-6 weeks post-injury. Paradoxically, the extent of locomotor recovery was impaired in the MRL/MpJ mice. Close examination of the chronic lesion site revealed evidence of ongoing degeneration both within and surrounding the lesion site. Thus, the regenerative genetic wound healing traits of the MRL/MpJ mice contribute to the evolution of a lesion environment that supports enhanced axon growth after SCI. However, this response occurs at the expense of meaningful functional recovery.

The effects of tubulation on healing and scar formation after transection of the adult rat spinal cord.

PURPOSE: The purpose of this study was to characterize the effects of implantation of a collagen tube on healing and scar formation following transection of tbc adult rat spinal cord. METHODS: The spinal cords of adult rats were completely transected at the mid-thoracic level. At 30 days after injury, the cellular and extra-cellular components of repair tissue present within tubulated and non-tubulated (control) wounds were compared using qualitative and quantitative histological techniques. RESULTS: The presence of the tube reduced fibrocollagenous scar invasion into the gap, promoted astrocyte migration, and oriented axonal and connective tissue components of the repair tissue. Tube implants supported the regeneration of a substantial number of myelinated axons. A notable finding was the identification of cells containing a contractile actin isoform in the healing spinal cord. CONCLUSIONS: The tubulation model allows for the study of spinal cord wound healing and axon elongation in a controlled experimental environment within the tube lumen. Using this model, it will be possible to study manipulation of the healing response by the introduction of exogenous agents within the tube.

Referred phantom sensations and cortical reorganization after spinal cord injury in humans.

To test the hypothesis that cortical remapping supports phantom sensations, we examined referred phantom sensations and cortical activation in humans after spinal-cord injury (SCI) at the thoracic level (T3-T12). Of 12 SCI subjects, 9 reported phantom sensations, and 2 reported referred phantom sensations. In both of these subjects, referred phantom sensations were evoked by contact in reference zones (RZ) that were not adjacent in the periphery and were not predicted to be adjacent in the postcentral gyrus (PoCG), suggesting that representations separated by centimeters of cortical space were simultaneously engaged. This finding was supported by functional MRI (fMRI). In a subject with a T6-level complete SCI, contact in RZ on the left or right forearm projected referred phantom sensations to the ipsilateral chest. During fMRI, contact in either forearm RZ evoked activity in the central PoCG (the position of the forearm representation) and the medial PoCG (the position of the chest representation) with >/=1.6 cm of nonresponsive cortex intervening. In contrast, stimulation in non-RZ forearm and palm regions in this subject and in lesion-matched SCI subjects evoked central but not medial PoCG activation. Our findings support a relation between PoCG activation and the percept of referred phantom sensations. These results, however, present an alternative to somatotopic cortical reorganization, namely, cortical plasticity expressed in coactivation of nonadjacent representations. The observed pattern suggests that somatotopic subcortical remapping, projected to the cortex, can support perceptual and cortical reorganization after deafferentation in humans.

Symptomatic gallstones in patients with spinal cord injury.

Patients with spinal cord injury (SCI) have an increased prevalence of cholelithiasis. The goal of this study was to clarify the presentation and management of symptomatic gallstone disease in patients with SCI. We performed a retrospective study of presentation of gallstone complications in patients with SCI who underwent cholecystectomy for complications of gallstone disease. The West Roxbury Veterans Administration Medical Center SCI registry (605 patients) was searched for patients who had undergone cholecystectomy more than 1 year after SCI (35 patients). Gallbladder disease profiles for the 35 patients undergoing cholecystectomy for complications of gallstone disease were prepared, including demographics, clinical presentation, diagnostic studies, operative and pathologic findings, and postoperative complications. All patients were white. Thirty-four were male and the mean age was 50 years (range 35 to 65 years). The majority of patients (66%) complained of right upper quadrant abdominal pain, even those patients with SCI at high (i.e., cervical) levels. Of the 35 patients in our study group, 22 (63%) had biliary colic and chronic cholecystitis, nine (26%) had acute cholecystitis (gangrenous cholecystitis in two), two (6%) had choledocholithiasis symptoms or cholangitis, and two (6%) had gallstone pancreatitis. Major perioperative morbidity occurred in two (6%) of the 35 patients (pulmonary embolus; intraoperative hemorrhage), and there were no deaths. In the great majority of patients with SCI, cholelithiasis presents with chronic pain and not with life-threatening complications. Our findings suggest that presentation is no more acute in patients with SCI than in the general population. Characteristic symptoms and signs are not necessarily obscured by SCI injury, regardless of the level.

Unusual expression of the Hu paraneoplastic antigen in the visual system.

The Hu paraneoplastic antigen is an RNA binding protein important in the development and maintenance of neurons. Hu has homology with the product of Elav, a gene that is essential for Drosophila visual system development. Using immunohistochemical staining, the Hu antigen was identified in most retinal neurons but was not detected in the photoreceptors. Hu is, therefore, not essential for the maintenance of all differentiated neurons. Hu immunolabeling was consistently found in glia in the optic nerve. The presence of the Hu antigen in a subset of glial cells emphasizes the heterogeneity of glial phenotype and raises the possibility that these cells may retain properties of undifferentiated neural stem cells.

Basic fibroblast growth factor increases nitric oxide synthase production in bovine endothelial cells.

Basic fibroblast growth factor (bFGF) and nitric oxide (NO) are expressed by endothelial cells (EC) and are involved in regulation of endothelial functions. In vivo, bFGF has a hypotensive effect which is mediated, in part, through activation of nitric oxide synthase (NOS) and the subsequent generation of NO. Thus we hypothesized that regulation of NOS in EC might be modulated by bFGF. bFGF treatment of EC in vitro resulted in increased NADPH diaphorase staining, a histochemical marker associated with the presence of NOS. Using cGMP generation in a reporter cell as a bioassay for NO release, we demonstrated that bFGF treatment of EC leads to increased production of biologically active NO. Furthermore, bFGF treatment of EC resulted in an increase in cellular content of the endothelial form of NOS as shown by Western blot analysis. Finally, Northern blot analysis was used to demonstrate that message levels of the constitutive, calcium-dependent, endothelial form of NOS is increased in EC by treatment with bFGF in vitro. These results suggest that bFGF has potential to regulate vascular tone through the modulation of levels of endothelial NOS.

Hypoxia inhibits expression of eNOS via transcriptional and posttranscriptional mechanisms.

Normal blood vessel tone is maintained by a balance of vasoconstrictors and vasodilators produced by endothelial cells in the vasculature. Nitric oxide (NO) is a potent vasodilator that causes vascular smooth muscle cell relaxation by elevating intracellular guanosine 3',5'-cyclic monophosphate (cGMP) levels. The physiological mechanisms regulating NO production in the vasculature are not completely understood. We report here that production of this vasodilator by vascular endothelial cells can be significantly suppressed by hypoxia. Exposing human endothelial cells to low PO2 results in 40-60% reduction in the steady-state mRNA levels of endothelial constitutive NO synthase (eNOS), the major enzyme responsible for NO production in these cells. The lower levels of eNOS mRNA result from decreased transcription of the gene as well as reduced message stability. In endothelial-smooth muscle cell co-culture experiments, hypoxic endothelial cells stimulated significantly less cGMP production by smooth muscle cells than the corresponding normoxic controls. This inhibitory effect of hypoxia on NOS production by endothelial cells occurs after 24 h of hypoxia and persists for at least 48 h. These new findings suggest that hypoxia might cause changes in blood vessel tone through compound mechanisms: by increasing the production of endothelium-derived vasoconstrictors and, as shown here, by suppressing the production of vasodilators like NO.

Optic nerve injury alters basic fibroblast growth factor localization in the retina and optic tract.

Basic fibroblast growth factor (bFGF) is thought to be a trophic factor for several classes of neurons. Its distribution changes in response to cortical neural injury. We have determined the effect of injury to the optic nerve on localization of bFGF in the rodent retina and visual pathways. Our observations were confirmed by using different antisera and monoclonal antibodies. While photoreceptors normally contain virtually no bFGF, crushing the optic nerve causes a striking increase, over a period of several weeks, in the amount of bFGF in retinal photoreceptors. Since photoreceptors do not synapse directly upon the injured ganglion cells, intermediary cells must participate in the cascade of events that results in the elevated bFGF. In light of the observation that exogenous bFGF protects photoreceptors from photodamage (Faktorovich et al., 1992), this increase in bFGF in photoreceptors may explain, in part, why crushing the optic nerve protects photorecptors against photodamage (Bush and Williams, 1991). Whereas bFGF is constitutively found in glia in the optic nerve, little bFGF is found in glia in the optic tract. However, damage to the optic nerve increases bFGF in astrocytes in the optic tract. This change occurs within days, suggesting that a relatively direct signal may intervene between the injured axon and the adjacent glial cells. Thus, despite the fact that the optic nerve and optic tract are contiguous structures through which axons of retinal ganglion cells project, the glial elements in these structures express distinct properties, because of differences in either glial subclasses or microenvironment.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxygen-induced retinopathy in the mouse.

PURPOSE: To develop oxygen-induced retinopathy in the mouse with reproducible and quantifiable proliferative retinal neovascularization suitable for examining pathogenesis and therapeutic intervention for retinal neovascularization in retinopathy of prematurity (ROP) and other vasculopathologies. METHODS: One-week-old C57BL/6J mice were exposed to 75% oxygen for 5 days and then to room air. A novel fluorescein-dextran perfusion method has been developed to assess the vascular pattern. The proliferative neovascular response was quantified by counting the nuclei of new vessels extending from the retina into the vitreous in 6 microns sagittal cross-sections. Cross-sections were also stained for glial fibrillary acidic protein (GFAP). RESULTS: Fluorescein-dextran angiography delineated the entire vascular pattern, including neovascular tufts in flat-mounted retinas. Hyperoxia-induced neovascularization occurred at the junction between the vascularized and avascular retina in the mid-periphery. Retinal neovascularization occurred in all the pups between postnatal day 17 and postnatal day 21. There was a mean of 89 neovascular nuclei per cross-section of 9 eyes in hyperoxia compared to less than 1 nucleus per cross-section of 8 eyes in the normoxia control (P < 0.0001). Proliferative vessels were not associated with GFAP-positive astrocyte processes. CONCLUSIONS: The authors have described a reproducible and quantifiable mouse model of oxygen-induced retinal neovascularization that should prove useful for the study of pathogenesis of retinal neovascularization as well as for the study of medical intervention for ROP and other retinal angiopathies.

Substance P immunoreactive astrocytes are present in multiple sclerosis plaques.

Substance P (SP)-like immunoreactive cells were identified in postmortem white matter tissue from patients with multiple sclerosis (MS). Labelled cells, which by morphologic criteria could be identified as astrocytes, were located at the edge of both active (e.g. inflammatory) and inactive (e.g. non-inflammatory) MS lesions. By contrast, SP-immunoreactive astrocytes were not found in normal controls and were only occasionally present in other conditions associated with astrogliosis. These data suggest that SP, a potent mediator of vasodilatation and local immune responses, may play a role in the genesis of the MS plaque. These results also extend the repertoire of potential interactions which may occur between astrocytes and cells of the immune system.

Neuronal organization underlying visually elicited prey orienting in the frog--I. Effects of various unilateral lesions.

We have studied the effects on frog orienting behavior of three lesions: unilateral optic nerve section, unilateral tectal lobe ablation, and unilateral transverse hemisection of the neuraxis at a level just caudal to the optic tectum. Unilateral optic nerve section and unilateral tectal lobe ablation produce very similar deficits in visually elicited responses to prey items, an absence of responses for stimuli at locations within the monocular field of one eye. Unilateral hemisection, in contrast, results in abnormalities in visually elicited responses over a wider area, encompassing the entire ipsilateral visual hemifield. The hemisection deficit also differs in character from that following optic nerve section or tectal lesion. Within the affected hemifield, frogs do not fail to respond to stimuli but rather respond with abnormally directed movements. The movements, regardless of stimulus eccentricity on the horizontal, are always forwardly directed. While not varying with horizontal eccentricity, the movements do vary with stimulus elevation and distance. The variation with stimulus distance in the affected hemifield is somewhat different from that in the opposite hemifield. We conclude from the behavior that remains after hemisection lesions that there must exist bilateral descending tectofugal paths capable of triggering movements which vary with stimulus elevation and distance, and a crossed descending tectofugal path capable of triggering turns into one visual hemifield. That the deficit area is larger following a hemisection than following tectal lobe ablation indicates that the hemisection has affected the ability of both tectal lobes to trigger turns in one direction. A possible interpretation of this finding is that the lesion has interrupted not only the crossed descending tectofugal path from one tectal lobe but an uncrossed descending tectofugal path from the other. This hypothetical pathway as well as the others mentioned is incorporated in a model of the organization of the post-tectal circuitry involved in orienting.

Neuronal organization underlying visually elicited prey orienting in the frog--II. Anatomical studies on the laterality of central projections.

A complete transverse hemisection of the neuraxis just caudal to the optic tectum in the frog, Rana pipiens, results in a failure to orient toward stimuli in one visual hemifield [Kostyk and Grobstein (1986) Neuroscience 21, 41-55]. The extent of the deficit area implies disturbances in the outputs triggered by both tectal lobes. In this paper we report studies aimed at determining more precisely what damage is involved in producing the hemisection deficit, with the broader objective of identifying particular neural structures which may be important in visually elicited orienting. Small lesions at the level of the hemisection which are restricted to the ventromedial white tracts result in an orienting deficit identical to that produced by a complete hemisection. Large lesions which spare the ventromedial white tracts are without significant effect on orienting turns. The finding is consistent with the hypothesis that the hemisection deficit results from interruption of tectal outflow paths. Interestingly, partial damage of the ventromedial white tracts does not result in disconnection of any local tectal region from premotor circuitry but instead systematically alters the turns triggered from all tectal regions. Ventrolateral lesions at the same level do not produce deficits in orienting but do disturb optokinetic behavior. Introduction of horseradish peroxidase into ventromedial lesions produces retrograde labeling in a large number of structures both rostral and caudal of the lesion. Labeling patterns following introduction of horseradish peroxidase into ventrolateral lesions, which do not affect orienting turns, were qualitatively similar but differed quantitatively. The observed patterns of tectal cell labeling make it unlikely that the hemisection deficit can be accounted for in terms of interruption of direct projections deriving from complementary regions of the two tectal lobes. They also indicate that if there exists an uncrossed tectal outflow adequate to trigger orienting turns, it must be by way of an indirect projection. A more general analysis of the labeling patterns suggests that a crossed tectal projection and uncrossed projections from three midbrain tegmental nuclei (the anterodorsal tegmental nucleus, the nucleus profunds lateralis and the nucleus of the medial longitudinal fasciculus) are likely to be involved in triggering orienting turns. The three midbrain tegmental nuclei are of particular interest in that they provide possible anatomical substrates for an indirect uncrossed descending tectal outflow path.

Neuronal organization underlying visually elicited prey orienting in the frog--III. Evidence for the existence of an uncrossed descending tectofugal pathway.

A complete transverse hemisection of the neuraxis just caudal to the optic tectum in the frog, Rana pipiens, results in a failure to orient toward stimuli in one visual hemifield [Kostyk and Grobstein (1986) Neuroscience 21, 41-55]. This finding indicates that each tectal lobe gives rise to a crossed descending pathway adequate to cause turns in a direction contralateral to that tectal lobe, and suggests that each may also give rise to an uncrossed descending pathway adequate to cause turns in the ipsilateral direction. To determine whether there is in fact such an uncrossed pathway, we have studied the orienting behavior of frogs after lesions which interrupt crossed pathways. Two groups of animals were studied. In one group we made midline lesions of the ansulate commissure, through which run the major crossed descending projections from both tectal lobes. In the other group, we combined a complete transverse hemisection with removal of the tectal lobe on the same side of the brain, leaving intact only an uncrossed pathway from one tectal lobe. A persistence of orienting turns was observed in both groups of animals. In both, the direction of the turns was that expected on the assumption that an uncrossed pathway would cause ipsilateral turns. We conclude that such a pathway exists. While both groups of animals turned in the expected directions, they did so for stimuli at unexpected locations. Increasingly eccentric stimulus locations to one side of the mid-sagittal plane were associated with increasing amplitude turns to the other. The observation suggests that tectal regions mapping areas of visual space to one side of the mid-sagittal plane are capable of triggering turns not only in that direction but in the opposite direction as well. In the case of ansulate commissure section, mirrored orienting responses were observed for tactile stimuli as well. These and other behavioral anomalies described in the preceding papers [Kostyk and Grobstein (1986) Neuroscience 21, 41-55 and 57-82] suggest that between the topographic retinotectal projection and the premotor circuitry for orienting there may exist an intermediate processing step, one in which stimulus location is represented in a generalized spatial coordinate frame.

Visual orienting deficits in frogs with various unilateral lesions.

We have studied the visual prey acquisition behavior of frogs with unilateral optic nerve section, unilateral tectal lobe ablation, and unilateral transverse hemisection at a level between the tectum and the cerebellum. The first two groups of animals oriented normally to stimuli throughout the region of visual field overlap and failed to respond to more peripheral stimuli on one side. Hemisected animals responded to stimuli at all positions in the visual field. For stimuli located contralateral to the lesion, the frogs oriented normally. For ipsilateral stimuli, the frogs oriented forward.

Orienting behavior of juvenile frogs with both a pre-metamorphically rotated and a normal eye.

We have studied the orienting behavior of juvenile Rana pipiens in which one eye was rotated at late larval stages and the other eye left intact. Such frogs orient accurately to stimuli falling solely in the visual field of the intact eye and systematically misorient to stimuli falling solely in the field of the rotated eye. Stimuli within the area of visual field overlap elicited two distinct sets of responses, one attributable to the normal and the other to the rotated eye.