ISSN 1916-6958

2010; Volume 1 (Number 2): Pages 72-75

ORIGINAL RESEARCH

Neuroprotective
effects of glycine in rats with permanent cerebral ischemia

Rebeca URIBE-ESCAMILLAa,
Krystell PADILLA-MARTÍNa, Angélica GONZÁLEZ-MACIELb, Emilio
ARCH-TIRADOa, Alejandro A. NAVA-OCAMPOc,d,  Alfonso
ALFARO-RODRÍGUEZa,*.

aLaboratory of Neurochemistry, National Institute
of Rehabilitation, Secretaría de Salud, México DF, Mexico, bLaboratory
of Electronic Microscopy, National Institute of Pediatrics, Secretaría de
Salud, México DF, Mexico, cDepartment of Pharmacology &
Toxicology, Faculty of Medicine, University of Toronto, Toronto ON, Canada, dPharmaReasons,
Toronto ON, Canada

*Corresponding author:
[email-masked]

ABSTRACT

Objective: The aim of this study was to evaluate the neuroprotective effects
of glycine in an experimental animal model of permanent brain ischemic injury.
Glycine is an inhibitory neurotransmitter amino acid that acts as
neuromodulator of N-methyl-D-aspartate (NMDA) receptors, critically involved in
the process of ischemic brain injury. Methods:
Twenty-four Wistar rats, 20 days old, were randomly allocated to one of the
following three groups. In Group 1: rats underwent a sham surgery where the
left carotid was visualized only; in Group 2, rats underwent permanent left
carotid occlusion and received treatment with placebo; and in Group 3, rats
received glycine i.p. (40 mMol kg-1 24 h-1) for 30 days post-unilateral carotid occlusion. Thirty days
post-surgery, the animals were sacrificed and brains were examined by
histopathology. Results: In Group 1,
normal brain histopathology was observed. In Group 2, tortuous capillary
vessels were observed in the brain in addition to necrotic areas in
hippocampus, medial vestibular nucleus, piriform and cerebral cortex, and
thalamus. In Group 3, histopathological alterations were restricted to
thalamus. Conclusions: Glycine
administered i.p. to rats with permanent left carotid occlusion limited the
ischemic brain damage probably by increasing the neurological availability of
glycine concentration enough to prevent the desensitization of NMDA receptors
and consequently altering the cascade of events that lead to cellular death.

Key words

Brain
ischemia; Glycine; Neuroprotective agents.

RÉSUMÉ

Objectif: Évaluation des effets neuroprotecteurs de la
glycine dans un modèle animal expérimental d’ischémie cérébrale permanente. La
glycine est un acide aminé neurotransmetteur inhibiteur qui agit comme neuromodulateur
des récepteurs au N-méthyl-D-aspartate (NMDA) impliqués dans le processus
d’ischémie cérébrale. Méthodes: Vingt-quatre
rats Wistar âgés de 20 jours ont été assignés at random à un des trois groupes suivants. Groupe 1: les rats ont
subi une chirurgie factice où la carotide gauche était uniquement visualisée;
Groupe 2: les rats ont subi une occlusion permanente de la carotide gauche et
ont reçu un placebo; Groupe 3: les rats ont reçu de la glycine i.p. (40 mMol kg-1
24 h-1) pendant 30 jours après l’occlusion carotidienne. Trente
jours après la chirurgie les animaux ont été sacrifiés et les cerveaux examinés
par histopathologie. Résultats:
Groupe 1: le cerveau a une histopathologie normale. Groupe 2: présence de
vaisseaux capillaires cérébraux tortueux ainsi que de zones de nécrose dans
l’hippocampe, le noyau vestibulaire médian, les cortex piriforme et cérébral,
et le thalamus. Groupe 3 : les altérations sont limitées au thalamus. Conclusions: La glycine administrée en
i.p. chez des rats avec occlusion permanente de la carotide gauche limite les
lésions ischémiques cérébrales probablement en augmentant sa disponibilité
neurologique en suffisance pour prévenir la désensibilisation des récepteurs au
NMDA et altérer la cascade qui aboutit à la mort cellulaire.

Mots clés

Ischémie
cérébrale; Glycine; Agents neuroprotecteurs

RESUMEN

Objetivo: Evaluar los efectos neuroprotectores de la
glicina en un modelo animal de daño cerebral por isquemia permanente. La
glicina es un aminoácido neurotransmisor inhibitorio que actúa como modulador
del receptor N-metil-D-aspartato receptor (NMDA), implicado en forma importante
en el proceso de isquemia cerebral. Métodos:
24 ratas Wistar de 20 días de edad fueron asignadas aleatoriamente a uno de
tres grupos. En el Grupo 1 las ratas fueron sometidas a una cirugía falsa donde
la carótida izquierda fue visualizada; en el Grupo 2 se realizó una oclusión
permanente de la arteria carótida izquierda y los animales recibieron placebo,
y en el Grupo 3 las ratas recibieron glicina i.p. (40 mMol kg-1 24 h-1)
durante 30 días posteriores a la cirugía. A los 30 días posteriores a la
cirugía, las ratas fueron sacrificadas y sus cerebros analizados por
histopatología. Resultados: En el
Grupo 1 se observó una histopatología normal del cerebro; en el Grupo 2 se
observaron vasos capilares tortuosos además de áreas necróticas en el
hipocampo, núcleo medio vestibular, la corteza periforme y cerebral, y tálamo.
En el Grupo 3, las modificaciones histopatologías fueron restringidas al tálamo.
Conclusiones: La administración de
glicina i.p. a ratas con oclusión permanente de la arteria carótida izquierda
limitó el daño secundario a isquemia cerebral, probablemente debido al
incremento de la concentración neurológica de glicina a niveles suficientes
como para prevenir la desensibilización de los receptores de NMDA, alterando la
cascada de acontecimientos que llevan a la muerte celular.

Palabras clave

Isquemia
cerebral; Glicina; Agentes neuroprotectores

INTRODUCTION

Ischemia of central nervous system is associated with
increased extracellular concentrations of glutamate, which leads to uncontrolled
activation of NMDA receptors and favors the influx of calcium. These changes
progress to mitochondrial and DNA damage, ion imbalance, cell membrane disruption
and, finally, cell death. As death cells release glutamate, this process can be
extended to neighboring cells [1].

Several experimental studies have shown that inhibition of
glutamate release may offer a neuroprotective alternative [2] [3]. Glycine is an inhibitory neurotransmitter in the
brain and spinal cord, and plays a critical role as a modulator of NMDA
receptors [4]. Furthermore, peripheral administration of glycine
produces a clear dose-dependent elevation of the levels of this amino acid in
both serum and cerebrospinal fluid (CSF) [5]. However, the effect of extracellular glycine
concentrations on ischemic injury remains controversial as there is evidence that
high levels of extracellular glycine are indeed associated to ischemic damage [4] [6].

In order to evaluate the neuroprotective effects of glycine in
an experimental animal model of brain ischemia, we assessed the histopathology
of brains obtained from rats treated for 30 days after unilateral carotid
occlusion.

METHODS

Animals

The study was approved by the Institutional Committee for
Animal Use at the National Institute of Rehabilitation, and rats were handled
according to the national guidelines in Mexico. Twenty-four Wistar rats,
weighing approximately 25 g
at the beginning of the study, were randomly allocated to three groups (n= 8
animals/group). Animals in Group 1 underwent a sham operation where the common
carotid artery (CCA) was isolated but not occluded. Occlusion of the CCA was
performed to animals in Groups 2 and 3. Rats were maintained on a 12 hour
light/dark cycle with unlimited access to food and water. Glycine
(Sigma-Aldrich Quimica SA de CV, Toluca, Estado de México, Mexico) was
dissolved in 0.9% normal saline and administered intraperitoneally (i.p.) to
animals in Group 3 at a dose of 40 mMol kg-1 day-1 for 30
days post-ligation whereas the animals in Groups 1 and 2 received saline at a
volume adjusted to simulate treatment with glycine.

Surgical procedure

The morning of the surgery, animals were anesthetized with
i.p. administration of ketamine (50 mg kg-1) and xylazine (5 mg kg-1).
The animals were placed in dorsal position and their extremities were fixed;
the skin was clamped and exposed by a gentle traction of the clamps; an
incision of approximately 2.5
cm long was performed in the mean anterior line of the
neck, at approximately 3 cm
distal to the sternum. Using the omohyoid and sternocleidomastoid muscles as
references, we located and dissected the vascular package. We identified the
internal jugular vein, the CCA (pulsating), and the vague nerve. The OCC was
dissected, occluded with silk suture and sectioned. For the purpose of
standardizing the surgical procedure, occlusion was always performed to the
left CCA. The sectioned segment was analyzed histopathologically in order to
confirm that the proper vessel was occluded. The sham operation was undertaken
in a similar manner, except CCA were not occluded or sectioned.

Histological evaluation

Thirty days after the surgical procedure, animals were
anesthetized as described above. A trans-cardiac perfusion through the left
ventricle was performed with formaldehyde 4% in a buffer of phosphates (pH
7.0). The brain was extracted and maintained in this solution for a period not
longer than a week. Thereafter, brains were embedded in paraffin and cut in
5-µm sections. Cresyl violet staining was used for the assessment of cell loss.
A total of 45 slices were prepared per brain.

RESULTS

As expected, in Group 1 (sham operation and treatment with
saline) the cerebral cortex, hippocampus (Figure 1 A
& D), thalamus and cerebellum (Figure 2 A, D &
G) showed normal morphology. In Group 2 (occlusion of CCA and treatment with
saline), the hemisphere contralateral to the occlusion showed histopathological
damage including capillary vessel dilation in all brain regions. There were
some hyperchromic cells in the cortex (Figure 1) and
dentate gyrus. In the hippocampus, we observed areas of dilated capillaries
with endothelium damage and necrotic granular cells. The thalamus was found
with some capillaries dilated and endothelial damage (Figure
2 B, E & H). In Group 3, most animals had brain alterations restricted
only to thalamic structures. In the hippocampus, very few dilated capillaries
were observed. Thalamus showed only few dilated capillaries with endothelial
damage (Figure 2 C). The cerebellum had normal
morphology (Figure 2 F & I).

Figure
1. Cerebral cortex (A, B, C; magnification × 200) and hippocampus (D, E, F; magnification
× 200) following 30 days of ischemia. Normal morphology was observed in the
sham-operation group (A & D). In Group 2 (occlusion-saline), necrotic
areas were observed in the cortex (arrow) & hippocampus (B & E). In
Group 3 (occlusion-glycine), hyperchromic cells were found in cortex and
dentate gyrus, hippocampus with dilated capillaries and damage in endothelium
(C & F). Slices were stained with cresyl violet.

Figure
2. Thalamus (A, B, C; ; magnification × 200) and cerebellum (D, E & F at
magnification × 100; and G, H & I at magnification × 200) of rats after
30 days of ischemia. Normal cellular
pattern was observed in Group 1 - sham operation (A,
D & G). In Group 2, groups of necrotic neurons were found in the thalamus
and cerebellum (B, E & H). In the glycine-treated group, only dilated
capillaries and some level of capillary endothelial damage (arrow) were
observed in thalamus whereas no alterations were appreciated in the cerebellum
(F & I).

DISCUSSION

The present study found that administration of glycine in an
animal model of brain ischemic injury prevented cell damage. Glycine is
normally found in the interstitial space at a brain concentration of 4 mol L-1
in the cortex [7] [8], and is a well-recognized major inhibitory
neurotransmitter in the spinal cord as well as playing an important role as a
co-agonist of NMDA subtype receptors [9] [10]. NMDA receptor has been implicated in a number of
processes related to neuronal development and plasticity as well as in neuronal
survival [11] [12]. Accordingly, it was previously found that exogenously
applied glycine facilitates the generation of long time potentiation (LTP) in
CA1 and dentate region of rat hippocampal slices by activating glycine
modulatory sites associated with NMDA receptors [10] [13]. The pyramidal neurons of hippocampus sector CA1 are
considered as the most vulnerable neurons to ischemia [14] [15]. Furthermore, injection of glycine and AFK 90-7 (a glycine
phosphorylated derivative) increased the survival of laboratory animals under
the conditions of ischemia caused by ligation of carotid artery [16]. Administration of peripheral glycine produced a
distinct rise in levels of glycine in serum and cerebrospinal fluid [5]. However, levels of glycine have been associated with
the severity of hypoxic encephalopathy-ischemic observed in human infants [17]. In addition, rats have a large capacity to recover,
and in many behavior tests the impairment in performance after focal cerebral
ischemia is transient [18]. Therefore, more studies are needed in order to
confirm the protective effects observed in our study. In this context, the
model of ischemia with permanent occlusion of CCA would be preferable over
models of transient ischemia for testing drug treatment of brain ischemic
damage.

CONCLUSION

Glycine administered i.p. to rats with permanent left carotid
occlusion limited ischemic brain damage, probably by increasing the
neurological availability of glycine concentration enough to prevent the
desensitization of NMDA receptors and consequently altering the cascade of
events that lead to cellular death.

AUTHORS’ PARTICIPATION

R U-E, E A-T and A A-R conceived the study and performed the
experiments. All the authors participated discussing the results and drafting
the manuscript.

CONFLICT OF INTERESTS/DISCLAIMERS

AA N-O is member of the Editorial Board of the journal.

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