Gemst: a taylor-made combination that reverts neuroanatomical changes in stroke

Abstract

In a single transient middle cerebral artery occlusion model of stroke and using immunohistochemical techniques, the effects of a new therapeutic approach named Gemst (a member of the Poly-L-Lysine innovative therapies) have been studied in the rat brain. The expression of inflammatory (CD45, CD11b), oxidative (NO-tryptophan, NO2-tyrosine) and indoleamine 2, 3-dioxygenase pathway (kynurenic acid, 3-hydroxy anthranilic acid) markers has been evaluated in early and late phases of stroke. For this purpose, we have developed eight highly specific monoclonal antibodies directed against some of these markers. In the early phase (3 and 5 days of the stroke, we observed no effect of Gemst treatment (7.5 mg/day, subcutaneously for 3, 5 days). In the late phase (21 days) of stroke and exclusively in the ipsilateral side of non-treated animals an overexpression of kynurenic acid, 3-hydroxy anthranilic acid, CD45, CD11b, GFAP and ionized calcium-binding adapter molecule 1 (IBA-1) was found. In treated animals, the overexpression of the four former markers was completely abolished whereas the overexpression of the two latter ones was decreased down to normal levels. Gemst reversed the pathological conditions of stroke to normal situations. Gemst exerts a multifunctional action: down-regulates the indoleamine 2, 3-dioxygenase pathway and abolishes brain infiltration, microglial activation and gliosis. Moreover, Gemst has no effect on the expression of doublecortin, a protein involved in neuronal migration. Gemst could be a new drug for the treatment of stroke since it reverses the pathological findings of stroke and normalizes brain tissue conditions following the ischemic insult.

Figure 1.

21D group. Immunoreactivity for IBA-1 (A, B), CD45 (C, D) and CD11b (E, F) in non-treated (NaCl, positive control) (A, C, E) and treated (Gemst) (B, D, F) animals. Contra: contralateral side; Ipsi: ipsilateral side. IBA-1 is overexpressed in the infarcted region (A), whereas the immunoreactivity observed in this region for CD45 (C) or CD11b (E) was completely abolished in the ipsilateral side of treated animals (D, F). Photographs were taken using a Leica microscope/Neurolucida software.

Figure 2.

21D group. Ipsilateral side. Immunoreactivity for IBA-1 (A-F) in non-treated (NaCl, positive control) (A, C, E) and treated (Gemst) (B, D, F) animals. Compared with Gemst treated animals, note the overexpression of IBA-1 in non-treated animals. E) Highpower magnification of the region delimited by the rectangle in C. F) High-power magnification of the region delimited by the rectangle in D. LV, lateral ventricle; M, medial; V, ventral.

Figure 3.

21D group. Ipsilateral side. Immunoreactivity for CD45 (A-D) in non-treated (NaCl, positive control) (A, C, D) and treated (Gemst) (B) animals. Note the absence of immunoreactivity in animals treated with Gemst (B). C) High-power magnification of the region delimited by the rectangle in A. D) High-power magnification of the region delimited by the rectangle in C. LV, lateral ventricle; M, medial; V, ventral.

Figure 4.

21D group. Ipsilateral side. Immunoreactivity for CD11b (A-D) in non-treated (NaCl, positive control) (A, C, D) and treated (Gemst) (B) animals. Note the absence of immunoreactivity in animals treated with Gemst (B). C) High-power magnification of the region delimited by the rectangle in A. D) High-power magnification of the region delimited by the rectangle in C. LV, lateral ventricle; M, medial; V, ventral.

Figure 5.

21D group. Ipsilateral side. Immunoreactivity for KYNA (A-D) in non-treated (NaCl, positive control) (A, C-D) and treated (Gemst) (B) animals. Note the absence of immunoreactivity in animals treated with Gemst (B). CC, cerebral cortex; M, medial; V, ventral.

Figure 6.

21D group. Ipsilateral side. Immunoreactivity for 3-HAA (A-F) in non-treated (NaCl, positive control) (A, C-F) and treated (Gemst) (B) animals. Note the absence of immunoreactivity in animals treated with Gemst (B). E) Inset at top left: a higher magnification of the region delimited by the rectangle. F) Inset at top left: higher magnification of the region delimited by the rectangle. LV, lateral ventricle; M, medial; V, ventral.

Figure 7.

21D group. Ipsilateral side. Double-labeling immunohistochemistry. The coexistence of KYNA (brown labelling) and 3-HAA (blue labelling, arrowheads) is shown in astrocytes.