Blockade of adrenoreceptors inhibits the splenic response to stroke

Abstract

Recent studies have highlighted the involvement of the peripheral immune system in delayed cellular degeneration after stroke. In the permanent middle cerebral artery occlusion (MCAO) model of stroke, the spleen decreases in size. This reduction occurs through the release of splenic immune cells. Systemic treatment with human umbilical cord blood cells (HUCBC) 24 h post-stroke blocks the reduction in spleen size while significantly reducing infarct volume. Splenectomy 2 weeks prior to MCAO also reduces infarct volume, further demonstrating the detrimental role of this organ in stroke-induced neurodegeneration. Activation of the sympathetic nervous system after MCAO results in elevated catecholamine levels both at the level of the spleen, through direct splenic innervation, and throughout the systemic circulation upon release from the adrenal medulla. These catecholamines bind to splenic alpha and beta adrenoreceptors. This study examines whether catecholamines regulate the splenic response to stroke. Male Sprague-Dawley rats either underwent splenic denervation 2 weeks prior to MCAO or received injections of carvedilol, a pan adrenergic receptor blocker, prazosin, an alpha1 receptor blocker, or propranolol, a beta receptor blocker. Denervation was confirmed by reduced splenic expression of tyrosine hydroxylase. Denervation prior to MCAO did not alter infarct volume or spleen size. Propranolol treatment also had no effects on these outcomes. Treatment with either prazosin or carvedilol prevented the reduction in spleen size, yet only carvedilol significantly reduced infarct volume (p < 0.05). These results demonstrate that circulating blood borne catecholamines regulate the splenic response to stroke through the activation of both alpha and beta adrenergic receptors.

Figure 1. Denervation has no effect on spleen shrinkage 48 hr post-MCAO

Spleens of rats subjected to denervation prior to stroke showed reduced weight when removed 48 hours after MCAO compared to sham-operated controls (*p<0.05). There was no significant difference in spleen weight of animals subjected to denervation-MCAO when compared to MCAO-only.

Figure 2. Tyrosine hydroxylase labeling 48 hr post-MCAO

TH immunoreactivity localized to nerve fibers (arrows) surrounding splenic blood vessels (BV) and was reduced in spleens from animals that underwent denervation (C) in contrast to labeled sections of animals subjected to sham-splenectomy (A). Spleens of rats subjected to MCAO-only (B) showed a similar labeling pattern as observed in sham-operated controls. Quantification (D) revealed a significant decrease in TH immunoreactivity in denervated spleens relative to spleens from animals subjected to sham-splenectomy or MCAO-only (*p<0.01).

Figure 3. Fluoro-Jade stains the infarct of denervated rats 48 hr post-MCAO

Fluoro-Jade staining shows a large area of damage in the ipsilateral hemisphere from brains of denervation-MCAO rats (C). Rats subjected to MCAO-only showed a similar neurodegenerative profile (B). Brain sections from sham-MCAO rats (A) and sham-MCAO+denervation animals showed no Fluoro-Jade labeled areas (not shown). Quantification (D) revealed no significant change in infarct volume between denervation-MCAO and MCAO-only rats, while splenectomy prior to stroke significantly reduced infarct volume (*p<0.01). Scale bar = 2 mm.

Figure 4. Thionin staining is reduced in denervated rats 48 hr post-MCAO

Thionin-stained Nissl Bodies were reduced in tissues from rats subjected to denervation-MCAO (C) and MCAO-only (B). Brain sections from sham-MCAO (A) and sham-MCAO+denervation (not shown) rats showed uniform thionin staining typical of an uninjured brain. Quantification (D) revealed no significant change in infarct volume between denervation-MCAO and MCAO-only rats, while splenectomy prior to stroke significantly reduced infarct volume (*p<0.01). Scale bar = 2 mm.

Figure 5. Short-term treatment with adrenoreceptor antagonists does not alter blood pressure

Blood pressures of rats were measured at 24 hours preoperative, 0 hours, and 24 hours postoperative to determine whether alterations occurred after short-term administration of adrenoreceptor antagonists. Quantification showed no significant differences in systolic or diastolic rates across treatment groups at any time point examined.

Figure 6. The effects of adrenoreceptor antagonists on spleen weight

Spleen weights from rats treated with vehicle, carvedilol, prazosin or propranolol were recorded and quantified. Results showed that spleen weights from carvedilol- and prazosin-treated rats were significantly greater than those from rats subjected to MCAO-only (*p<0.05).

Figure 7. The effects of adrenoreceptor antagonists on infarct volume

No Fluoro-Jade staining was present in brain sections from sham-MCAO rats (A). Rats that received carvedilol prior to stroke showed reduced Fluoro-Jade staining (C) relative to rats subjected to MCAO-only (B). Treatment with prazosin (D) or propranolol (E) produced a staining profile similar to that observed in MCAO-only rats. Quantification of infarct volume (F) revealed a significant reduction in rats treated with carvedilol compared to prazosin, propranolol, or MCAO-only (*p<0.05). Scale bar = 2 mm.

Figure 8. Adrenoreceptor antagonists do not alter splenic immune cell counts

Flow cytometry was performed on spleen samples collected from rats that were subjected to either sham-MCAO or MCAO with administration of adrenoreceptor antagonists. Quantification showed no significant alterations in the immune cell types present within the splenocyte fractions across treatment groups, indicating that neither prazosin, propranolol, or carvedilol altered the relative abundance of immune cell types present within the spleen.

Figure 9. The effects of adrenoreceptor antagonists on inflammatory cytokine levels

Levels of the inflammatory cytokines IL-1ϐ and TNF-α were quantified from spleens of rats treated with carvedilol, prazosin or propranolol 48 hours after MCAO. Of all the adrenergic receptor blockers tested, only prazosin decreased TNF-α levels in the spleen relative to MCAO-only or sham-operated rats (A) (*p<0.05). In contrast, only propranolol treatment increased IL-1ϐ expression after MCAO compared to the splenic expression from MCAO- or sham-operated rats (B) (*p<0.05). Carvedilol treatment had no effect on the expression of these cytokines.