Brain protection using autologous bone marrow cell, metalloproteinase inhibitors, and metabolic treatment in cerebral ischemia

Abstract

Despite advances in imaging, understanding the underlying pathways, and clinical translation of animal models of disease there remains an urgent need for therapies that reduce brain damage after stroke and promote functional recovery in patients. Blocking oxidant radicals, reducing matrix metalloproteinase-induced neuronal damage, and use of stem cell therapy have been proposed and tested individually in prior studies. Here we provide a comprehensive integrative management approach to reducing damage and promoting recovery by combining biological therapies targeting these areas. In a rat model of transient cerebral ischemia (middle cerebral artery occlusion) gene delivery vectors were used to overexpress tissue inhibitor of matrix metalloproteinase 1 and 2 (TIMP1 and TIMP2) 3 days before ischemia. After occlusion, autologous bone marrow cells alone or in combination with agents to improve NO bioavailability were administered intraarterially. When infarct size, BrdU incorporation, and motor function recovery were determined in the treatment groups the largest beneficial effect was seen in rats receiving the triple combined therapy, surpassing effects of single or double therapies. Our study highlights the utility of combined drug, gene, and cell therapy in the treatment of stroke.

Fig. 1.

The treatment groups subjected to MCAO and reperfusion (45 min later) at day 0. Rats were pretreated with AdTIMP1/TIMP2, l-arginine/vitamin E, or a combination of these. Autologous BMCs were infused 6 h after MCAO and reperfusion. Animals were killed 14 days after MCAO for histologic studies. Group 1 (control) containing rats receiving only MCAO and rats receiving MCAO + control vector virus. Subsequently, for clarity, these groups in Figs. 3–5 and Tables 1 and 2 were kept separately and named Group (MCAO alone) and Group 2 (MCAO + control vector virus).

Fig. 2.

Infarct volume values (TTC) in six groups. ∗, P < 0.05 vs. group 1; ∗∗, P < 0.01 vs. group 1 and P < 0.05 vs. group 2; ○, P < 0.001 vs. group 1 and P < 0.01 vs. groups 2 and 3; ○○, P < 0.05 vs. group 1; §, NS vs. group 1.

Fig. 3.

Brain effects of joint treatments. (A) RT-PCR for TIMP1 and TIMP2 at 1, 2, and 3 days after transgene delivery. (B) Immunostaining for BrdU on coronal sections. BrdU⁺ cells were detected in the ipsilateral cortex near the infarct boundary (B1 and B2) and subventricular area (B3 and B4). (C) Cumulative BrdU staining in the groups. ∗, P < 0.001 vs. groups 1, 2, 6, and 7; ∗∗, P < 0.0005 vs. groups 1, 2, 6, and 7; §, P < 0.05 vs. group 3. Group 1 (control), only MCAO; group 3, autologous BMCs injected 6 h after MCAO; group 4, BMCs in association with AdTIMP1/TIMP2 pretreatment; group 5, BMCs in association with AdTIMP1/TIMP2 and concurrent treatment (120 mg/kg l-arginine i.p. per day and 2.4 mg/kg vitamin E per day); group 6, MCAO in association with AdTIMP1/TIMP2. Group 7 received MCAO with the metabolic treatment. (D) Representative double-immunofluorescent staining (merged) in ischemic brains under laser-scanning confocal microscopy of the BrdU⁺ cells (green) coupled to expression of GFAP (red), VWF (red), MAP-2 (red), and Neu-N (red).

Fig. 4.

Vessel perimeter and density. VWF immunostaining shows enlarged thin wall vessels (vessel perimeter and density) along cortical IBZ in the BMC-treated group (group 3) or in combination with TIMPs (group 4) and metabolic supplementation (group 5). ∗, P < 0.05 compared with groups 1, 2, 6, and 7; §, P < 0.05 vs. group 3.

Fig. 5.

Motor function recovery. A repeated-measures ANOVA revealed that a significant interaction was found among treatments and time (days) for duration on rod. A Mann–Whitney U test revealed that treated rats with BMCs and TIMPs with and without metabolic treatment showed significant improvement in neurological outcome. Group 1 (control), only MCAO; group 2 (control), MCAO and control vector virus; group 3, autologous BMCs injected 6 h after MCAO; group 4, BMCs in association with AdTIMP1/TIMP2 pretreatment; group 5, BMCs in association with AdTIMP1/TIMP2 and concurrent metabolic treatment (120 mg/kg l-arginine i.p. per day and 2.4 mg/kg vitamin E per day; group 6, MCAO in association with AdTIMP1/TIMP2; group 7 received MCAO with the metabolic treatment. ∗, P < 0.05 vs. groups 1 and 2; #, P < 0.01 vs. groups 1 and 2; +, P < 0.05 vs. group 3; §, P < 0.05 vs. groups 3 and 4.