Neuroprotection by endogenous and exogenous PACAP following stroke

Abstract

We investigated the effects of PACAP treatment, and endogenous PACAP deficiency, on infarct volume, neurological function, and the cerebrocortical transcriptional response in a mouse model of stroke, middle cerebral artery occlusion (MCAO). PACAP-38 administered i.v. or i.c.v. 1 h after MCAO significantly reduced infarct volume, and ameliorated functional motor deficits measured 24 h later in wild-type mice. Infarct volumes and neurological deficits (walking faults) were both greater in PACAP-deficient than in wild-type mice, but treatment with PACAP reduced lesion volume and neurological deficits in PACAP-deficient mice to the same level of improvement as in wild-type mice. A 35,546-clone mouse cDNA microarray was used to investigate cortical transcriptional changes associated with cerebral ischemia in wild-type and PACAP-deficient mice, and with PACAP treatment after MCAO in wild-type mice. 229 known (named) transcripts were increased (228) or decreased (1) in abundance at least 50% following cerebral ischemia in wild-type mice. 49 transcripts were significantly up-regulated only at 1 h post-MCAO (acute response transcripts), 142 were up-regulated only at 24 h post-MCAO (delayed response transcripts) and 37 transcripts were up-regulated at both times (sustained response transcripts). More than half of these are transcripts not previously reported to be altered in ischemia. A larger percentage of genes up-regulated at 24 hr than at 1 hr required endogenous PACAP, suggesting a more prominent role for PACAP in later response to injury than in the initial response. This is consistent with a neuroprotective role for PACAP in late response to injury, i.e., even when administered 1 hr or more after MCAO. Putative injury effector transcripts regulated by PACAP include beta-actin, midline 2, and metallothionein 1. Potential neuroprotective transcripts include several demonstrated to be PACAP-regulated in other contexts. Prominent among these were transcripts encoding the PACAP-regulated gene Ier3, and the neuropeptides enkephalin, substance P (tachykinin 1), and neurotensin.

Fig. 1

MCAO in the mouse. A. Experimental flowchart of the cerebral ischemia protocol. Animals were subjected to MCAO or sham surgery at 0 hr; PACAP or saline was administered at 1 hr; neurological evaluations and microarray analyses were performed at 1 hr and 24 hr; infarct volume was assessed at 24 hr after MCAO. B. Coronal sections of brain slices fixed with formaldehyde and stained with cresyl violet, showing the effects of PACAP (i.v. injection, 1 hr post-MCAO) on infarct volume in both wild type and PACAP-deficient mice 24 h following MCAO. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Effect of endogenous PACAP and PACAP treatment on neurological and neuropathological outcomes after MCAO. A. Initial neurological severity score at 1 hr, n = 8), B. Change in neurological severity score at 24 hr (*: p < 0.01 compared to corresponding untreated group, n = 8), C. Change in number of walking faults from 1 to 24 hr (#: p < 0.05 compared to the untreated wild-type group; : p < 0.05 compared to the corresponding untreated control group, n = 7), and D. Infarct volume at 24 hr (#: p < 0.05, compared to untreated wild type mice, n = 7). Blue bars, MCAO alone; red bars, MCAO with i.v. PACAP; yellow bars, MCAO with i.c.v. PACAP. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Changes in the cerebrocortical transcriptome 1 and 24 h after MCAO. Venn diagram depicts transcripts up-regulated at 1 and 24 h post-MCAO, and divides transcripts into those up-regulated only at 1 h (acute), at both 1 and 24 h (sustained) and only at 24 h (delayed). Named transcripts, i.e. those with designated gene name and associated gene i.d. (LocusLink) designation, in each category are listed in Tables 1–3.