Docosahexaenoic acid complexed to albumin provides neuroprotection after experimental stroke in aged rats

Abstract

Recently we have shown that docosahexaenoic acid complexed to albumin (DHA-Alb) is neuroprotective after experimental stroke in young rats. The purpose of this study was to determine whether treatment with DHA-Alb would be protective in aged rats after focal cerebral ischemia. Isoflurane/nitrous oxide-anesthetized normothermic (brain temperature 36-36.5°C) Sprague-Dawley aged rats (18-months old) received 2h middle cerebral artery occlusion (MCAo) by poly-l-lysine-coated intraluminal suture. The neurological status was evaluated during occlusion (60min) and on days 1, 2, 3 and 7 after MCAo; a grading scale of 0-12 was employed. DHA (5mg/kg), Alb (0.63g/kg), DHA-Alb (5mg/kg+0.63g/kg) or saline was administered i.v. 3h after onset of stroke (n=8-10 per group). Ex vivo T2-weighted imaging (T2WI) of the brains was conducted on an 11.7T MRI on day 7 and 3D reconstructions were generated. Infarct volumes and number of GFAP (reactive astrocytes), ED-1 (activated microglia/microphages), NeuN (neurons)-positive cells and SMI-71 (positive vessels) were counted in the cortex and striatum at the level of the central lesion. Physiological variables were entirely comparable between groups. Animals treated with DHA-Alb showed significantly improved neurological scores compared to vehicle rats; 33% improvement on day 1; 39% on day 2; 41% on day 3; and 45% on day 7. Total and cortical lesion volumes computed from T2WI were significantly reduced by DHA-Alb treatment (62 and 69%, respectively). In addition, treatment with DHA-Alb reduced cortical and total brain infarction while promoting cell survival. We conclude that DHA-Alb therapy is highly neuroprotective in aged rats following focal cerebral ischemia and has potential for the effective treatment of ischemic stroke in aged individuals.

Keywords: Behavior; Histopathology; Magnetic resonance imaging; Middle cerebral artery occlusion; Rat; Stroke.

Figure 1

Total neurological score (normal score=0, maximal score=12) during MCAo (60 min) and on days 1, 2, 3 and 7 after treatment. All treatments were administered intravenously at 3 h after onset of stroke. DHA and DHA-Alb treatment significantly improved neurological score compared to the saline group during the 7-day survival period. In addition, DHA-Alb treatment significantly improved the neurological score compared to the corresponding Alb group on day 7. Values shown are means ± SEM. #P<0.05 versus saline group; *P<0.05 DHA-Alb versus Alb group (repeated measures ANOVA followed by Bonferroni tests).

Figure 2. Ex vivo MRI at 7d after MCAo

Panel A: Representative T2WI from saline, DHA-, Alb- and DHA-Alb-treated rats. T2 hyperintensities were observed in the cortex and striatum of saline-treated rats, consistent with ongoing edema formation. DHA- and Alb-treated animals had smaller lesion size, with only partial cortex and subcortical involvement. In contrast, DHA-Alb-treated rats had primarily only a small subcortical lesion. Panel B: 3D reconstructions of MRI-derived lesion volumes from high resolution T2 weighted images (T2WI). Saline-treated rats showed large cortical and subcortical lesion volumes. Rats treated with DHA or Alb show less lesion volume, mostly in the subcortical area. By contrast, lesion volume was dramatically reduced in rats treated with DHA-Alb and was mostly localized in the subcortex. Panel C: Cortical, subcortical and total lesion volumes were computed from T2WI. Only treatment with DHA-Alb significantly reduced cortical, subcortical and total lesion volumes compared to the saline-treated group. In addition, DHA-Alb therapy reduced cortical and total lesion volumes compared to the corresponding Alb group. DHA and Alb treatments alone also reduced lesion volumes, but were not significantly different from saline and from each other. Values shown are means ± SEM., #P<0.05 versus saline group; *P<0.05 DHA-Alb versus Alb group (repeated measures ANOVA followed by Bonferroni tests).

Figure 3. Histopathology at 7d after MCAo

Panel A: Computer-generated MosaiX processed images (Zeiss Axio Imager.M1; AxioVision Release 4.6.3) of Nissl stained paraffin-embedded brain sections from rats treated with saline, DHA, Alb or DHA-Alb. Saline-treated rats showed large cortical and subcortical infarction. Rats treated with DHA or Alb show less extensive damage, mostly in the subcortical area. In contrast, DHA-Alb treated rat shows a very small infarction, involving only subcortical region. Panel B: Cortical, subcortical and total infarct volumes in four groups. Total infarct volume was corrected for brain swelling. DHA-Alb treatment reduced cortical and total infarct volumes compared to corresponding Alb group. DHA and Alb treatment alone also reduced infarct volumes, but were not significantly different from saline and from each other. Values shown are means ± SEM., *P<0.05 #P<0.05 versus saline group; *P<0.05 DHA-Alb versus Alb group (repeated measures ANOVA followed by Bonferroni tests).

Figure 4

Cell count for GFAP positive astrocytes, NeuN positive neurons, SMI-71 positive vessels and ED1 positive microglia cells on day 7 after 2h of MCAo. Coronal brain diagram showing locations of regions for cell count (bregma level - 0.3 mm) in cortex (A, B, and C) and striatum (S). Alb- and DHA-Alb-treated rats expressed increased numbers of GFAP compared to the saline-treated group (Panel A). All treatments (DHA, Alb, DHA-Alb) increased numbers of NeuN positive cells and SMI-71 immunopositive vessels compared to saline-treated rats (Panels B and C). In addition, DHA-Alb treatment increased SMI-71 positive vessels compared to the corresponding Alb group (Panel C). DHA, Alb, and DHA-Alb treatments decreased microglial invasion of the infarcted tissue while salinetreated rats showed significant microglial infiltration of the infarct regions (Panel D). In addition, DHA-Alb treatment decreased ED1 positive microglia compared to the corresponding Alb group (Panel D). Values shown are means ± SEM., *P<0.05 #P<0.05 versus saline group; *P<0.05 DHA-Alb versus Alb group (repeated measures ANOVA followed by Bonferroni tests).