Institute of Fundamental Technological Research

An excessive glutamate level can result in excitotoxic damage and death of central nervous system (CNS) cells, and is involved in the pathogenesis of many CNS diseases. It may also be related to a failure of the blood-spinal cord barrier (BSCB). This study was aimed at examining the effects of extended administration of monosodium glutamate on the BSCB and spinal cord cells in adult male Wistar rats. The glutamate was delivered by subarachnoidal application of glutamate-carrying electrospun nanofiber mat dressing at the lumbar enlargement level. Half of the rats with the glutamate-loaded mat application were treated systemically with the histone deacetylase inhibitor valproic acid. A group of intact rats and a rat group with subarachnoidal application of an ‘empty’ (i.e., carrying no glutamate) nanofiber mat dressing served as controls. All the rats were euthanized three weeks later and lumbar fragments of their spinal cords were harvested for histological, immunohistochemical and ultrastructural studies. The samples from controls revealed normal parenchyma and BSCB morphology, whereas those from rats with the glutamate-loaded nanofiber mat dressing showed many intraparenchymal microhemorrhages of variable sizes. The capillaries in the vicinity of the glutamate-carrying dressing (in the meninges and white matter alike) were edematous and leaky, and their endothelial cells showed degenerative changes: extensive swelling, enhanced vacuo­lization and the presence of vascular intraluminal projections. However, endothelial tight junctions were generally well preserved. Some endothelial cells were dying by necrosis or apoptosis. The adjacent parenchyma showed astrogliosis with astrocytic hypertrophy and swelling of perivascular astrocytic feet. Neurons in the parenchyma revealed multiple symptoms of degeneration, including, inter alia, perikaryal, dendritic and axonal swelling, and destruction of organelles. All the damage symptoms were slightly less severe in the rats given valproic acid treatment, and were absent from both the intact rats and the rats with ‘empty’ nanofiber mat dressing. These results demonstrate that glutamate-loaded nanofiber mat dressing can locally create glutamate levels capable of damaging BSCB and that the resulting damage can be mitigated with concurrent systemic valproate treatment.

astrocyte, blood-spinal cord barrier, CNS damage, degeneration, endothelium, excitotoxicity, glutamate, neuron, valproate, vessels

(Sub)chronic local drug application is clearly superior to systemic administration, but may be associated with substantial obstacles, particularly regarding the applications to highly sensitive central nervous system (CNS) structures that are shielded from the outer environment by the blood-brain barrier. Violation of the integrity of the barrier and CNS tissues by a permanently implanted probe or cannula meant for prolonged administration of drugs into specific CNS structures can be a severe confounding factor because of the resulting inflammatory reactions. In this study, we tested the utility of a novel way for (sub)chronic local delivery of highly active (i.e., used in very low amounts) drugs to the rat spinal cord employing a non-woven nanofiber mat dressing. To this end, we compared the morphology and motoneuron ( + ) counts in spinal cord cervical and lumbar segments between rats with glutamate-loaded nanofiber mats applied to the lumbar enlargement and rats with analogical implants carrying no glutamate. Half of the rats with glutamate-loaded implants were given daily valproate treatment to test its potential for counteracting the detrimental effects of glutamate excess. The mats were prepared in-house by electrospinning of an emulsion made of a solution of the biocompatible and biodegradable poly(L-lactide-co-caprolactone) polymer in a mixture of organic solvents, an aqueous phase with or without monosodium glutamate, and sodium dodecyl sulfate as an emulsifier; the final glutamate content was 1.4 µg/mg of the mat. Three weeks after mat implantation there was no inflammation or considerable damage of the spinal cord motoneuron population in the rats with the subarachnoid dressing of a glutamate-free mat, whereas the spinal cords of the rats with glutamate-loaded nanofiber mats showed clear symptoms of excitotoxic damage and a substantial increase in dying/damaged motoneuron numbers in both segments studied. The rats given systemic valproate treatment showed significantly lower percentages of damaged/dying motoneurons in their lumbar enlargements. These results demonstrate the capacity of nanofiber mats for generation of neurotoxic glutamate in the rat CNS. However, the tested nanofiber mats need further improvements aimed at extending the period of effective drug release and rendering the release more steady.

CNS injury, electrospinning, excitotoxicity, glutamate, motoneuron, nanofibers, neurodegeneration, spinal cord, valproate

Introduction:
Introduction of nanotechnology into medicine has provided new therapeutic options. It has been demonstrated that implantation of nanofiber mats after nervous system injury allowed to diminish scar size and infl ammatory reaction. It is also possible that, due to their ability to release active factors, nanofiber mats may replace intracerebral probes. To assess potential usefulness of nanofiber mats in releasing active substances we implanted them into the spinal cord subarachnoid space in adult Wistar rats.

Material and Method:
The experimental animals were divided into four groups: group 1 – rats with implanted nanofiber mats, group 2 – rats with implanted nanofiber mats releasing glutamate, group 3 – rats with nanofiber mats releasing glutamate and treated orally by sodium valproate, and group 4 – control animals without nanofiber mats. The animals were killed 21 days after the mats implantation. Then, histopathological, immunohistochemical and ultrastructural evaluation of the spinal cords was performed.

Results:
Morphological assessment revealed that implantation of nanofi ber mats caused neither spinal cord damage nor inflammation (group 1). Also nanofi ber mats releasing glutamate did not produce inflammatory reaction (group 2 and 3) although in group 2 morphological changes indicating toxic influence of glutamate were observed. These changes were less severe in group 3.

Conclusions:
(1) Nanofi ber mats are biocompatible and can be useful in long-term animal experiments. (2) Nanofi ber mats are able to release glutamate into the subarachnoid space. (3) Sodium valproate has a protective influence against glutamate toxicity.

electrospinning, nanofibers, drug delivery, neurology, sodium glutamate, animal model