An energy theory of glaucoma.

A radial array of fortified astrocytes (FASTs) is the load bearing structure of the rat optic nerve head (ONH). At the retinal end the ONH is suspended on a fluid filled extracellular space occupied by modified pigment cells which generate a glomerular-like formation of villi. We propose that regulation of fluid in and out of this space may contribute to buffering the normal fluctuations of intraocular pressure. The energy requirement for the fluid transfer process is provided by the dense vascularity of the ONH and is reflected in the giant mitochondria of the FASTs. We propose that glaucoma occurs when a maintained rise in pressure overwhelms the capacity of this regulatory system. Under these circumstances the FAST array becomes detached from its anchorage in the surrounding ONH sheath. Progressively driven backwards by the pressure, the FASTs degenerate. We propose that the degeneration of the FASTs is associated with ischemic damage caused by the backward stretching of their blood supply. Retraction of the FAST processes deprives the retinal ganglion cell axons of their energy support, resulting in axotomy. We consider that our previously observed rescue of axons and FASTs by transplantation of olfactory ensheathing cells is due to replacement of this lost energy source.

Obtaining the olfactory bulb as a source of olfactory ensheathing cells with the use of minimally invasive neuroendoscopy-assisted supraorbital keyhole approach--cadaveric feasibility study.

BACKGROUND: Obtaining the human olfactory bulb (OB) for treatment of spinal cord injuries with olfactory ensheathing cells (OECs) requires the elaboration of a surgical approach that could meet the criteria of safety and minimal invasiveness. The aim of the study was to evaluate the suitability of the keyhole supraorbital craniotomy (SOC) with an eyebrow incision for obtaining OB for therapeutic purposes. METHODS: Seventeen SOCs were performed on nine fresh adult cadavers. The procedure of obtaining OB was conducted by neuroendoscope-assisted microsurgical dissection. Technical features related to the procedure were measured and adverse events were noted. The virtual three-dimensional planning was applied in six cases to verify the authorial A-C scale published previously. RESULTS: The intact OB was obtained in 10 (59%) cases and a minor injury was discovered in another 5 (29%) cases. In 2 (12%) specimens OB was severely damaged which was correlated with the minor neural tissue injury (Fi(2) = 0.44). While no case of an evident vascular injury was noted, there were 3 (18%) incidents of unintended frontal sinus opening positively correlated with the craniotomy width (Fi(2) = 0.44). The unfavorable three-dimensional (3D) configuration of the olfactory groove area was revealed in 66% of cases and highly correlated with OB injury (Fi(2) = 1.0) but not damage. CONCLUSIONS: The SOC via an eyebrow incision may be safely and effectively applied to obtain the OB as a source of OECs in roughly 90% of cases. Virtual 3D planning is useful in preoperative qualification of potential donors.

The supraorbital keyhole approach via an eyebrow incision applied to obtain the olfactory bulb as a source of olfactory ensheathing cells--radiological feasibility study.

BACKGROUND. The purpose of the study was to test the suitability of supraorbital keyhole craniotomy with an eyebrow incision for obtaining olfactory bulb for therapeutic purposes. METHODS. Fifty three high-resolution axial head computed tomography images of patients with a mean age of 55 ± 15 years were used. The exclusion criterion was a pathology of the anterior skull base. The virtual keyhole supraorbital craniotomy was placed on each side of a three-dimensional skull model with respect to the anatomical landmarks. Trajectories of neurosurgical instrument transitions to the anterior and posterior aspects of olfactory grooves (OGs) were subsequently designed with the use of a neuronavigation planning station and measured with correction allowing the avoidance of collisions with skull base structures. Three types of anatomical configuration were divided, reflecting the extent of the correction needed to reach the bottom of OG. RESULTS. Simulation of the surgical access and consequent metrological analysis was performed on 97 skull sides - 9 (8.5%) sides were excluded due to the large frontal sinus. The mean length of the craniotomy basis was 30.71 mm, lengths of the anterior and posterior trajectories were 53.25 and 58.24 mm, respectively (p < 0.0001). In 37% of cases the value of the corrections exceeded the depth of OG. CONCLUSIONS. The supraorbital keyhole approach via an eyebrow incision may be applied to obtain the olfactory bulb as a source of olfactory ensheathing cells in over 60% of cases. Further verification and evaluation of the surgical handiness based on cadaver specimens is justifiable.

[Studies on glial isomerization of lamina cribrosa in rat].

OBJECTIVE: To explore the mechanism of optic nerve damage in glaucoma by study on structure of glial lamina cribrosa(LC) in rats. METHODS: Experimental study. Albino Swiss(AS) rats were divided into 3 groups. Bilateral eyes of 10 normal rats were employed to be group I (right eye ) and group II (left eye) . Group III was from the left eyes of 13 rats underwent artificially intraocular hypertension in the right eyes. All rats were perfused and fixed with electronic microscopy fixative (2% paraformaldehyde +2% glutaraldehyde). Trimmed optic nerves were embedded with resin. Serial 1.5 µm thick 'semithin' sections were cut, either (2 eyes from group III) longitudinally, through the optic nerve head (ONH) from the retinal end to the commencement of the optic nerve, or (31 eyes) transversely (cross-sections). Ultrathin sections were cut in the middle of glial LC. The morphological observation of glial LC was obtained by light microscopy and transmission electron microscopy. Bonferroni correction was used to counteract the multiple comparison of each group. RESULTS: Fortified astrocytes formed the main supportive structure of glial LC in all rats, including group I, group II and group III. Astrocytes were ranked as a fan-like radial array, firmly attached ventrally to the sheath of the LC by thick basal processes, but dividing dorsally into progressively more slender processes with only delicate attachments to the sheath. These fortified astrocytes form ventral stout basal end feet, radial array, axon free-'preterminal' layer before terminating in a complex layer of fine interdigitating delicate branches at the dorsal. LC astrocytes were highly and uniformly electron dense throughout all the cell processes. An equally striking feature of the astrocytic processes was their massive cytoskeletal 'strengthening' of longitudinal massed filaments and tubules. Especially, massive filaments accumulated as cytoskeletal cores to form 'scaffold' of fortified astrocytes. There was vulnerable area in the dorsal of glial LC. This vulnerable area was isomerisation in bilateral eyes and different rats. There was different space in the vulnerable area. These space could be divided into 3 grades, (-), (+) and (++) . The number of (-), (+) and (++)were 1, 6, 3 eyes in group I, 1, 5, 4 eyes in group II, 1, 7, 3 eyes in group III. The Kruskal-Wallis test was used for statistical evaluations. There was no statistical differences of the ratio of (-), (+) and (++) in group I, group II and group III(χ(2) = 3.35, P = 0.187>0.05;group I vs group II, Z = -1.048, P = 0.294;group I vs group III Z = -1.691, P = 0.091;group II vs group III,Z = -1.343, P = 0.179). The ratio of space (-)was significantly less than space (+) and space (++) in group I, group II and group III(χ(2) = 23.88, P < 0.05; (-) vs (+) , Z = -2.821, P = 0.005; (-) vs (++) , Z = -2.726, P = 0.006). The ratio of space (+)was much more than space (++) in group I, group II and group III(Z = -4.410, P < 0.05). CONCLUSION: Glial isomerisation in LC may play a key role in glaucomatous optic nerve damage.

Structural basis of glaucoma: the fortified astrocytes of the optic nerve head are the target of raised intraocular pressure.

Increased intraocular pressure (IOP) damages the retinal ganglion cell axons as they pass through the optic nerve head (ONH). The massive connective tissue structure of the human lamina cribrosa is generally assumed to be the pressure transducer responsible for the damage. The rat, however, with no lamina cribrosa, suffers the same glaucomatous response to raised IOP. Here, we show that the astrocytes of the rat ONH are "fortified" by extraordinarily dense cytoskeletal filaments that would make them ideal transducers of distorting mechanical forces. The ONH astrocytes are arranged as a fan-like radial array, firmly attached ventrally to the sheath of the ONH by thick basal processes, but dividing dorsally into progressively more slender processes with only delicate attachments to the sheath. At 1 week after raising the IOP by an injection of magnetic microspheres into the anterior eye chamber, the fine dorsal processes of the ONH astrocytes are torn away from the surrounding sheath. There is no indication of distortion or compression of the axons. Subsequently, despite return of the IOP toward normal levels, the damage to the ONH progresses ventrally through the astrocytic cell bodies, resulting in complete loss of the fortified astrocytes and of the majority of the axons by around 4 weeks. We propose that the dorsal attachments of the astrocytes are the site of initial damage in glaucoma, and that the damage to the axons is not mechanical, but is a consequence oflocalized loss of metabolic support from the astrocytes (Tsacopoulos and Magistretti (1996) J Neurosci 16:877-885).

Role of the lesion scar in the response to damage and repair of the central nervous system.

Traumatic damage to the central nervous system (CNS) destroys the blood-brain barrier (BBB) and provokes the invasion of hematogenous cells into the neural tissue. Invading leukocytes, macrophages and lymphocytes secrete various cytokines that induce an inflammatory reaction in the injured CNS and result in local neural degeneration, formation of a cystic cavity and activation of glial cells around the lesion site. As a consequence of these processes, two types of scarring tissue are formed in the lesion site. One is a glial scar that consists in reactive astrocytes, reactive microglia and glial precursor cells. The other is a fibrotic scar formed by fibroblasts, which have invaded the lesion site from adjacent meningeal and perivascular cells. At the interface, the reactive astrocytes and the fibroblasts interact to form an organized tissue, the glia limitans. The astrocytic reaction has a protective role by reconstituting the BBB, preventing neuronal degeneration and limiting the spread of damage. While much attention has been paid to the inhibitory effects of the astrocytic component of the scars on axon regeneration, this review will cover a number of recent studies in which manipulations of the fibroblastic component of the scar by reagents, such as blockers of collagen synthesis have been found to be beneficial for axon regeneration. To what extent these changes in the fibroblasts act via subsequent downstream actions on the astrocytes remains for future investigation.

Comparison of bulbar and mucosal olfactory ensheathing cells using FACS and simultaneous antigenic bivariate cell cycle analysis.

Transplantation of olfactory ensheathing cells (OECs) is a promising route for CNS repair. There have, however, been major discrepancies between the results from different groups. Part of this can be attributed to variations in cell sources and culture protocols. Accurate estimation of the proportions of OECs and their associated fibroblasts (ONFs) and their evolution with time in culture is an essential baseline for establishing the reparative properties of transplants. In this study, we compare the evolution of cultures from the superficial layers of the olfactory bulb with tissue from the olfactory mucosa, both whole and split into lamina propria and epithelial layer. We used FACS based on p75 and Thy1 to provide a robust and objective numerical estimate of the numbers of OECs and ONFs, respectively in the cultures. A novel four color simultaneous antigenic bivariate cell cycle analysis shows that proliferation of OECs is time-limited, and is unable to prevent an overall loss of OECs with time. Overall, the numbers of OECs in the cultures were inversely correlated with the deposition of fibronectin (FN). Further, culture of the cells purified by flow cytometry shows that, whereas the Thy1 population is terminally differentiated, the p75 population from the mucosal samples generates subpopulations with different antigenic phenotypes, including the reappearance of a subpopulation of p75 cells expressing FN. Culturing epithelial samples at high density reveals an unexpected transient stem cell-like population of rapidly proliferating p75 positive cells.

An in vitro model of the inhibition of axon growth in the lesion scar formed after central nervous system injury.

After central nervous system (CNS) injury, meningeal fibroblasts migrate in the lesion center to form a fibrotic scar which is surrounded by end feet of reactive astrocytes. The fibrotic scar expresses various axonal growth-inhibitory molecules and creates a major impediment for axonal regeneration. We developed an in vitro model of the scar using coculture of cerebral astrocytes and meningeal fibroblasts by adding transforming growth factor-beta1 (TGF-beta1), a potent fibrogenic factor. Addition of TGF-beta1 to this coculture resulted in enhanced proliferation of fibroblasts and the formation of cell clusters which consisted of fibroblasts inside and surrounded by astrocytes. The cell cluster in culture densely accumulated the extracellular matrix molecules and axonal growth-inhibitory molecules similar to the fibrotic scar, and remarkably inhibited the neurite outgrowth of cerebellar neurons. Therefore, this culture system can be available to analyze the inhibitory property in the lesion site of CNS.

Novel strategies in brachial plexus repair after traumatic avulsion.

Clinical trials in spinal cord injury (SCI) can be affected by many confounding variables including spontaneous recovery, variation in the lesion type and extend. However, the clinical need and the paucity of effective therapies has spawned a large number of animal studies and clinical trials for SCI. In this review, we suggest that brachial plexus avulsion injury, a longitudinal spinal cord lesion, is a simpler model to test methods of spinal cord repair. We explore reconstructive techniques currently explored for the repair of brachial plexus avulsion and focus on the use of olfactory ensheathing cell transplantation as an adjunct treatment in brachial plexus repair.

Organotypic slice co-cultures reveal that early postnatal hippocampal axons lose the ability to grow along the fimbria, while retaining the ability to invade and arborise in septal neuropil.

The failure of cut axons to grow along fibre tracts in the adult CNS contrasts with their ability to do so in development. Organotypic slices culture of a number of areas enables the time of failure to be pinpointed to around the second week of postnatal life in the rat. 'Heterochronic' co-culture of slices above and below this age shows that the failure is due to the inability of the older axons to grow into either the same age or younger targets. Using hippocampo-septal slices the present experiments show that this failure is due to an inability to recognise the glial pathway of the fimbria, even when this is of a younger age. However, the older hippocampal neurons retain the ability to grow axons into septal target tissue when they are placed in direct contact with it. This exactly mirrors the inability of cut central axons to regenerate along their previous fibre pathways while they retain their ability to reinnervate neuropil.

Restoration of hand function in a rat model of repair of brachial plexus injury.

The incurability of spinal cord injury and subcortical strokes is due to the inability of nerve fibres to regenerate. One of the clearest clinical situations where failure of regeneration leads to a permanent functional deficit is avulsion of the brachial plexus. In current practice, surgical re-implantation of avulsed spinal roots provides a degree of motor recovery, but the patients neither recover sensation nor the use of the hand. In the present rat study, we show that transplantation of cultured adult olfactory ensheathing cells restores the sensory input needed for a complex, goal-directed fore-paw function and re-establishes synaptic transmission to the spinal grey matter and cuneate nucleus by providing a bridge for regeneration of severed dorsal root fibres into the spinal cord. Success in a first application of human olfactory ensheathing cells in clinical brachial plexus injury would open the way to the wider field of brain and spinal cord injuries.

Transplantation of cultured olfactory mucosal cells rescues optic nerve axons in a rat glaucoma model.

PURPOSE: To determine whether transplantation of olfactory mucosal cells (OMCs) is able to rescue the loss of optic nerve axons after the intraocular pressure (IOP) is elevated in rats. METHODS: The IOP was raised by injection of magnetic microspheres into the anterior chamber of the eye. OMCs cultured from the adult olfactory mucosa were transplanted into the region of the optic disc. RESULTS: We demonstrated that although the raised IOP returned to its normal level at six weeks, there was an irreversible 58% loss of optic nerve axons in the control group. However, the loss of the axons was reduced to 23% in the group with the transplanted OMCs. The Pattern Electroretinograms (pERG) showed that the decrement of the voltage amplitudes in association with the raised IOP was significantly alleviated in the group with transplantation of OMC. CONCLUSIONS: Transplantation of OMCs is able to rescue loss of optic nerve axons induced by raised IOP in the rats. The pERG recording suggested that the functional activities of the axons are also protected. TRANSLATIONAL RELEVANCE: The results demonstrated the ability of the transplanted OMCs to protect against the loss of the optic nerve axons and the loss of function caused by raised IOPs. The findings provide a basis for future human clinical trials by autografting OMCs from autologous nasal epithelial biopsies to treat or delay glaucoma diseases.

Regeneration of nigrostriatal dopaminergic axons after transplantation of olfactory ensheathing cells and fibroblasts prevents fibrotic scar formation at the lesion site.

The fibrotic scar formed after central nervous system injury has been considered an obstacle to axonal regeneration. The present study was designed to examine whether cell transplantation into a damaged central nervous system can reduce fibrotic scar formation and promote axonal regeneration. Nigrostriatal dopaminergic axons were unilaterally transected in rats and cultures of olfactory-ensheathing cells (OECs), and olfactory nerve fibroblasts were transplanted into the lesion site. In the absence of transplants, few tyrosine hydroxylase-immunoreactive axons extended across the lesion 2 weeks after the transection. Reactive astrocytes increased around the lesion, and a fibrotic scar containing type IV collagen deposits developed in the lesion center. The immunoreactivity of chondroitin sulfate side chains and core protein of NG2 proteoglycan increased in and around the lesion. One and 2 weeks after transection and simultaneous transplantation, dopaminergic axons regenerated across the transplanted tissues, which consisted of p75-immunoreactive OECs and fibronectin-immunoreactive fibroblasts. Reactive astrocytes and chondroitin sulfate immunoreactivity increased around the transplants, whereas the deposition of type IV collagen and fibrotic scar formation were completely prevented at the lesion site. Transplantation of meningeal fibroblasts similarly prevented the formation of the fibrotic scar, although its effect on regeneration was less potent than transplantation of OECs and olfactory nerve fibroblasts. The present results suggest that elimination of the inhibitory fibrotic scar is important for neural regeneration.

Partial Recovery of Proprioception in Rats with Dorsal Root Injury after Human Olfactory Bulb Cell Transplantation.

Transplanted human olfactory ensheathing cells (hOECs) were mixed with collagen into a unilateral transection of four dorsal roots (C6-T1) in a rat model. By mixing with collagen, the limited numbers of hOEC were maximized from an olfactory bulb biopsy and optimize cavity filling. Cyclosporine was administered daily to prevent immune rejection. Forelimb proprioception was assessed weekly in a vertical climb task. Half of the rats receiving hOEC transplants showed some functional improvement ("responders") over six weeks of the study while the other half did not ("nonresponders") and performed similarly to "injured only" rats. Transplanted cells were seen at both one week and six weeks after the surgical procedure; many were concentrated within the lesion cavity, but others were found with elongated processes in the overlying connective tissue. There were some fibers in the injury area associated with transplanted cells that were immunostained for neurofilament and TUJ1. Responder and nonresponder rats were compared with regard to microglial activation within the deep dorsal horn of cervical levels C7, C8 and also axon loss within the cuneate fasciculus at cervical level C3. Little difference was seen in microglial activation or axonal loss that could account for the improved proprioception in the responders group. This preliminary study is the first to transplant human olfactory bulb cells into a rat model of dorsal root injury; by refining each component part of the procedure, the repair potential of OECs can be maximized in a clinical setting.

Repair of spinal cord injury by transplantation of olfactory ensheathing cells.

Repair of spinal cord injury requires that severed axons are able to regenerate. Regrowth of axons is impeded by the loss of astrocytic pathways caused at the time of injury. Ensheathing glial cells cultured from the adult olfactory system can be transplanted into lesions and mediate both regeneration of axons and recovery of function.

Transplantation of Cultured Olfactory Bulb Cells Prevents Abnormal Sensory Responses During Recovery From Dorsal Root Avulsion in the Rat.

The central branches of the C7 and C8 dorsal roots were avulsed close to their entry point into the spinal cord in adult rats. The forepaw responses to heat and cold stimuli were tested at 1, 2, and 3 weeks after injury. Over this period, the paws were sensitive to both stimuli at 1-2 weeks and returned toward normal at 3 weeks. Immunohistology showed no evidence of axonal regeneration into the spinal cord in a control group of rats with avulsion only, implying that adjacent dorsal roots and their corresponding dermatomes were involved in the recovery. In a further group of rats, a mixture of bulbar olfactory ensheathing cells and olfactory nerve fibroblasts were transplanted into the gap between the avulsed roots and the spinal cord at the time of avulsion. These rats showed no evidence of either loss of sensation or exaggerated responses to stimuli at any of the time points from 1 to 3 weeks. Immunohistology showed that the transplanted cells formed a complete bridge, and the central branches of the dorsal root fibers had regenerated into the dorsal horn of the spinal cord. These regenerating axons, including Tuj1 and CGRP immunoreactive fibers, were ensheathed by the olfactory ensheathing cells. This confirms our previous demonstration of central regeneration by these transplants and suggests that such transplants may provide a useful means to prevent the development of abnormal sensations such as allodynia after spinal root lesions.

Olfactory ensheathing cells: ripples of an incoming tide?

Until now, brain and spinal cord injuries that sever nerve fibres have resulted in a degree of incurable functional loss. An incoming tide of research is now beginning to challenge this as yet unbreached sea wall. One of the most promising approaches involves a recently discovered type of cell, the olfactory ensheathing cell, which can be obtained from the adult nasal lining. In animal models transplantation of cultured olfactory ensheathing cells into an injured spinal cord induces regeneration, remyelination of severed spinal nerve fibres, and functional recovery. Although several clinical centres worldwide have shown an interest in applying this approach to patients with spinal cord injury, there is no agreement on cell technology, and claims of beneficial results lack independent confirmation. Important aspects still need to be worked out at the laboratory level. Overall, the outlook is optimistic, but there is still some way to go.

Astrocytic role in synapse formation after injury.

In 1969 a paper entitled Neuronal plasticity in the septal nuclei of the adult rat proposed that new synapses are formed in the adult brain after injury (Raisman, 1969). The quantitative electron microscopic study of the timed responses to selective partial denervation of the neuropil of the adult rat septal nuclei after distant transection of the hippocampal efferent axons in the fimbria showed that the new synapses arise by sprouting of surviving adjacent synapses which selectively take over the previously denervated sites and thus restore the number of synapses to normal. This article presents the evidence for the role of perisynaptic astrocytic processes in the removal and formation of synapses and considers its significance as one of the three major divisions of the astrocytic surface in terms of the axonal responses to injury and regeneration. This article is part of a Special Issue entitled SI:50th Anniversary Issue.

Functional Repair of Rat Corticospinal Tract Lesions Does Not Require Permanent Survival of an Immunoincompatible Transplant.

Cell transplantation is one of the most promising strategies for repair of human spinal cord injuries. Animal studies from a number of laboratories have shown that transplantation of olfactory ensheathing cells cultured from biopsies of the olfactory bulb mediate axonal regeneration and remyelination and restore lost functions in spinal cord injuries. For translation from small laboratory experimental injuries to the large spinal cord injuries encountered in human patients the numbers of cells that can be obtained from a patient's own olfactory bulb becomes a serious limiting factor. Furthermore, removal of an olfactory bulb requires invasive surgery and risks unilateral anosmia. We here report that xenografted mouse bulbar olfactory ensheathing cells immunoprotected by daily cyclosporine restore directed forepaw reaching function in rats with chronic C1/2 unilateral corticospinal tract lesions. Once function had been established for 10 days, cyclosporine was withdrawn. Thirteen out of 13 rats continued to increase directed forepaw reaching. Immunohistochemistry shows that in all cases neurofilament-positive axons were present in the lesion, but that the grafted cells had been totally rejected. This implies that once grafted cells have acted as bridges for axon regeneration across the lesion site their continued presence is no longer necessary for maintaining the restored function. This raises the possibility that in the future a protocol of temporary immunoprotection might allow for the use of the larger available numbers of immunoincompatible allografted cells or cell lines, which would avoid the need for removing a patient's olfactory bulb.

Transplantation of olfactory ensheathing cells into spinal cord lesions restores breathing and climbing.

One of the most devastating effects of damage to the upper spinal cord is the loss of the ability to breathe; patients suffering these injuries can be kept alive only with assisted ventilation. No known method for repairing these injuries exists. We report here the return of supraspinal control of breathing and major improvements in climbing after the application of a novel endogenous matrix transfer method. This method permits efficient transfer and retention of cultured adult rat olfactory ensheathing cells when transplanted into large lesions that destroy all tracts on one side at the upper cervical level of the adult rat spinal cord. This demonstrates that transplantation can produce simultaneous repair of two independent spinal functions.