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Folia Neuropathologica
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Original paper

Nanofiber mat spinal cord dressing-released glutamate impairs blood-spinal cord barrier

Dorota Sulejczak, Anna Taraszewska, Stanisław J. Chrapusta, Dorota Dziewulska, Paweł Nakielski, Janina Rafałowska

Folia Neuropathol 2016; 54 (4): 392-404
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Introduction

The blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB) are composite structures the role of which is selective separation of the cerebral and spinal parenchyma from the contents of the blood vessel; these barriers play both a protective and regulatory role for the respective central nervous system (CNS) parenchyma [3]. The blood-brain barrier and BSCB are prone to damage from insults of varying origin, including local inflammatory processes, ischemia, hypoxia, mechanical trauma, and more.
The two barriers slightly differ structurally and functionally, mostly in ultrastructure of their endothelial cells [8,12,32,34,40]. These differences may be responsible for diverse susceptibility of BBB and BSCB to some pathological conditions [3]. Many authors suggest that disruption of BSCB or alteration of its permeability may be of key importance for the consequences of acute injury of the spinal cord [47] and for the development of some CNS pathologies, such as multiple sclerosis [37], amyotrophic lateral sclerosis [15,29], spinal cord ischemia [16,18,27], and neuropathic pain [4].
The main components of BSCB are endothelial cells, basement membrane and pericytes; some role is also played by perivascular astrocytes, the processes of which envelop small blood vessels [3,9]. An important role of astrocytes in the CNS is to support neurons under both normal physiological and pathological conditions, both by metabolic and regulatory means [10,25,44,46]. Due to the presence of active uptake and/or metabolic systems for some monoamine and amino acid neurotransmitters they can also contribute to the regulation of their interstitial levels [19,20,39]. After various CNS insults, astroglial cells invade the damaged region, become activated and hypertrophic, and, inter alia, start to express and release multiple trophic factors, including chemokines and cytokines [13].
An excessive glutamate level can cause damage and death of CNS cells and is also known as an im­portant contributing factor in many CNS disorders [21,33]. Glutamate-damaged neurons show definite morphological alterations. Excitotoxic neuronal injury may correlate with BBB damage [7].
There is an ongoing search for new approaches to delivering various drugs into the CNS, and in particular those capable of chronic delivery. Our earlier data have shown the usefulness of subarachnoidal electrospun nanofiber mat dressing as the drug delivery system capable of long-term release of glutamate levels sufficient for damaging spinal cord motoneurons in the rat [36]. The aim of the present investigation was to examine the effects of this treatment on morphology of the various components of the BSCB, especially on endothelial cells.

Material and methods

Nanofiber mats

Drug-free (‘empty’) electrospun nanofiber mats as well as monosodium glutamate (MSG)-loaded electrospun nanofiber mats were prepared as described earlier [36].

Animals and experimental design

Adult male Wistar rats (starting body weight 250-300 g, n = 12) from the animal facility of the Mossakowski Medical Research Centre were used for the study. The rats were kept in three per opaque plastic cage (55 × 33 cm floor size) in an air-conditioned (60-70% relative humidity, 21 ± 2°C) room at a 12 h light/12 h dark day cycle (lights on at 7 a.m.), and were given free access to sterilized standard laboratory rat maintenance chow (Ssniff, Soest, Germany) and tap water.
The rats were randomized between four experimental groups of three rats each: 1) intact controls (group I), 2) rats with drug-free nanofiber mat application into the spinal cord subarachnoid space at the lumbar enlargement level (group II), 3) rats with MSG-loaded nanofiber mat (14.2 mg MSG/mg of the mat) into the subarachnoid space (group III); and 4) rats subjected to a surgical placement of the same glutamate-loaded mat into the subarachnoid space, which were additionally given by gavage one dose of the histone deacetylase inhibitor sodium valproate (Convulex syrup, 50 mg/ml, Gerot Pharmazeutika, Wien, Austria) daily, beginning on the surgery day (group IV). The initial valproate dose was 33.3 mg/kg body weight; the dosage was elevated by 8.3 mg/kg each day for 4 days and then was kept at 67 mg/kg body weight for the remainder of the study period.
Subarachnoidal implantation of nanofiber mat pieces (5 × 5 mm size) into rat spinal cords (at the lumbar enlargement level) was performed as described earlier [1]. Three weeks after the surgery, all rats were deeply anesthetized with pentobarbital (80 mg/kg, i.p.) and decapitated. The L1-L6 segments of their spinal cords were harvested and immediately fixed in 4% formaldehyde solution in PBS and next embedded in paraffin by a standard procedure. The paraffin blocks were then cut and processed for histological and immunohistochemical methods.
All animal use procedures were in agreement with the European Communities Council Directive on the protection of laboratory animals (86/609/EEC) and with the current Polish law. All efforts were made to reduce animal discomfort and the number of rats used. The protocol of the study has been accepted by the 4th Local Animal Experimentation Ethics Committee at the National Medicines Institute, Warsaw, Poland (Certificate No. 43/210).

Histology and immunohistochemistry

The paraffin-embedded spinal cord samples were cut crosswise into 8 µm sections. After deparaffinization and rehydration in a water-ethanol solution series by standard procedures, some of the sections were stained with hematoxylin-eosin mixture solution using a routine procedure. The sections selected for immunohistochemistry were first incubated with rabbit polyclonal anti-GFAP antibody (Dako cat. no. Z0334; diluted 1 : 4000). Next, the sections were incubated with goat anti-rabbit antibody (Beckman Coulter Inc., France, cat. no. IM0830; dil. 1 : 100) and then with streptavidin-horseradish peroxidase solution (Beckman Coulter cat. no. IM0309; dil. 1 : 500). The resulting immune complexes were visualized by a routine procedure with diaminobenzidine as the chromogen and then counterstained with hematoxylin. The intensity of GFAP immunostaining was assessed by an experimenter blinded to the samples identity, using a light microscope (Nikon, Japan) equipped with a CCD camera and a PC-based image analyzer system. Specificity of the staining was tested by running the same procedure on the respective sister sections with the primary antibody absent from the incubation mixture; the control sections revealed no immunosignal.

Transmission electron microscopy

Samples of lumbar spinal cord meant for electron microscopy were instantly fixed in a formaldehyde-glutaraldehyde (2%/2.5%) solution in cacodylate buffer pH 7.4 for 4 h. Next, they were cut into smaller fragments, rinsed in the cacodylate buffer, post-fixed in 1.0% OsO4 solution for 1 h, dehydrated by a standard procedure, embedded in epon resin, and cut into 40 µm-thick sections. The sections were stained with 1% toluidine blue and examined in a light microscope for block selection. The selected blocks were then cut into ultrathin sections that were stained with uranyl acetate and lead citrate and then examined with a model JEOL 1200EX (Jeol, Japan) electron microscope by an experimenter blinded to the samples identity.

Results

Light microscopy morphological and immunohistochemistry studies

Spinal cords from the intact rats and rats carrying ‘empty’ nanofiber mat dressing showed normal parenchyma and BSCB morphology. Spinal cords from the rats carrying MSG-loaded spinal cord dressing (group III) revealed the presence of multiple intraparenchymal microhemorrhages of varying sizes (Figs. 1 and 2). The capillaries in the vicinity of the dressing (both in the meninges and the white matter) were leaky and showed considerable swelling; there was no difference in these characteristics between rats with and without systemic valproic acid treatment (group IV and group III, respectively).
Immunohistochemistry revealed astrogliosis, pre­sence of numerous activated hypertrophic astrocytes (Fig. 3) and swelling of the astroglial end-feet pro­cesses located close to the leaky vessels both in group III and group IV; however, the symptoms were more severe in group III. No signs of astroglial reaction were found in the control group with ‘empty’ subarachnoidal nanofiber mat dressing (Fig. 3).

Electron microscopy studies

In the intact controls (group I) and in the rats with ‘empty’ nanofiber mat dressing (group II) (Fig. 4), electron microscopy showed proper morphology of blood vessels, neurons and glial cells. The structure of microvascular basement membrane was homogenous and tight (Figs. 2-4); the vicinity of the vessels showed the presence of normal-looking astrocytic end-feet and scanty intercellular space.
In group III rats, neurons revealed both cytoplasmic and mitochondrial edema, organelle decay, endoplasmic reticulum atrophy and cytoplasmic microvacuolization. These alterations were more extensive in postsynaptic dendrites (Fig. 5A) than in neuronal perikarya (Fig. 5B). Similar changes were seen in the cytoplasm of both astroglial cells (Fig. 5C), small vessel endothelial cells and pericytes (Fig. 5D). The vessels showed endothelial cells damage of varying intensity and thickened basement membrane; edema of perivascular end-feet processes and neuropil, and expansion of the interstitial space were apparent as well. Some vessels showed endothelial cells necrosis with destruction of cell membrane and deterioration of tight junctions (Figs. 5B and 6A). A typical pattern of apoptotic changes of the endothelium was but rarely seen (Fig. 6B). More frequently, the endothe­lium showed a mixed pattern of overlapping apoptotic and necrotic changes (Fig. 6C-D), with increased cytoplasm density and relatively well-preserved organelles in some (shrunken) cells, and cytoplasmic edema and vacuolization, organelle and cell membrane decay in the other cells. Most of the damaged endothelial cells had an irregular luminal surface, with multiple intraluminal cytoplasmic microvilli-like protrusions, increased numbers of plasmalemmal vesicles, cytoplasmic vacuoles and irregular endoplasmic reticulum channels (Fig. 6C-E). Most endothelial tight junctions remained closed (Fig. 6E); opened tight junctions were occasionally seen, especially in the proximity of the basement membrane. The basement membrane of the vessels was mostly thickened and stratified, with embedded fragments of pericytes (Fig. 6E). The adjacent parenchyma showed considerable edema and decay of neuropil and astrocytic perivascular processes (Fig. 6C-D).
In the spinal cords of group IV rats, the edema of the endothelium and perivascular astrocytic end-feet was of clearly lesser severity (Figs. 7A-B); the endothelial cells showed nearly normal luminal cellular membrane (with only occasional intraluminal protrusions), mostly well-preserved mitochondria, ribosomes and endoplasmic reticulum; single autophagic vacuoles were but sporadically seen. In summary, all the MSG-loaded electrospun nanofiber mat dressing-induced alterations were more severe in group III than in group IV rats.

Discussion

Glutamate belongs to the most extensively studied neurotransmitters. Its physiological content in the nervous tissue corresponds with the metabolic demand of the cells, and the BBB and BSCB protect CNS parenchyma from an influx of excessive extra-CNS glutamate amounts [26]. It is common knowledge that CNS glutamate concentration is greatly elevated in many CNS pathologies [23,35,43,48]. It has been demonstrated as well that the considerable ischemia-related elevation of intracerebral glutamate results in BBB damage and brain edema [41]. Glutamate is also assigned an important role in the pathomechanisms of many CNS pathologies, e.g., of epilepsy, ischemia and amyotrophic lateral sclerosis, and in acute brain or spinal cord injury [2,26,28,30].
The present study is a continuation of our earlier investigation that demonstrated a toxic effect of exogenous glutamate delivered by subarachnoidal application of MSG-loaded electrospun nanofiber mat dressing on spinal cord motoneurons and a neuroprotective action of concurrent sodium valproate treatment [36]. This study was aimed at determining the potential of subarachnoidal electrospun nanofiber mat dressing for delivering MSG in the amount sufficient for inducing BSCB damage, and at defining the effect of the exogenous glutamate on perivascular cells of the spinal cord parenchyma. We have demonstrated that the chronic action of the mat-released glutamate damages BSCB. Blood vessels become dilated and permeabilized, and their vicinity showed the presence of edema and multiple microhemorrhages. Our studies have also revealed a strong astrocytic reaction in the vicinity of the damaged blood vessels, which demonstrated itself in marked astrogliosis and cell activation. However, some of the astrocytes showed clasmatodendrosis evidencing cell degeneration. Electron microscopy has also demonstrated the presence of ‘empty’ astrocytic end-feet processes evidencing perivascular edema. Considerable alterations were also visible in the cytoplasm and organelles of endothelial cells and in the adjacent basement membrane. The vicinity of the vessels showed also the presence of degenerating neurons. Interestingly, the glutamate-evoked alterations of the BSCB did not significantly affect the state of these cells in that their condition, while poor, was not worse than that of the neurons more distal to the damaged vessels [36]. It is believed that the basis of the glutamate-induced cell damage are, inter alia, an enhanced generation of free radicals and excessive calcium ions levels [11,13,14,17,38,45], which ultimately cause permeabilization of blood vessel walls by damaging BBB/BSCB integrity via a variety of signal transduction pathways and/or by direct action on endothelial tight junctions [6].
Glutamate excitotoxicity causes severe disturbances in protein and lipid metabolism by activating AMPA, NMDA and kainate glutamate receptors [24,26], and in particular the endothelial NMDA receptor subset [5,22]. Excessive stimulation of the NMDA receptors causes imbalance of Na+ ions across plasma membranes [31], increased influx of Ca2+ ions and activation of a wide spectrum of intracellular enzymes, including kinases, proteases and phospholipases [26], and promotes free radical-related oxidative stress in endothelial cells [42]. The latter in turn causes activation of NMDA receptors, resulting in forming of a metabolic vicious circle that intensifies functional and structural disturbances of cell membranes and increases BBB/BSCB permeability.
All said, the results of this study indicate that MSG-loaded nanofiber mat dressing can create glutamate concentrations capable of damaging both the BSCB and the neighboring parenchyma in the rat. Whereas the glutamate released from the dressing damages them both considerably, the concurrent systemic administration of the histone deacetylase inhibitor valproic acid alleviates the detrimental consequences of the long-term action of excessive glutamate levels on parenchyma more than those seen in the barrier.

Acknowledgments

This work was supported by grant no. NN 401 014640 from the Ministry of Science and Higher Education of Poland, and by statutory funds from the Mossakowski Medical Research Centre, Polish Academy of Sciences. The authors thank Dr. Tomasz Ko­walczyk of the Institute of Fundamental Technological Research, Polish Academy of Sciences, for his consulting the preparation and in vitro characterization of the electrospun nanofiber mats.

Disclosure

Authors report no conflict of interest.

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Copyright: © 2016 Mossakowski Medical Research Centre Polish Academy of Sciences and the Polish Association of Neuropathologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0) License (http://creativecommons.org/licenses/by-nc-sa/4.0/), allowing third parties to copy and redistribute the material in any medium or format and to remix, transform, and build upon the material, provided the original work is properly cited and states its license.
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