Brain changes associated with thromboxane receptor antagonist SQ 29,548 treatment in a mouse model

Abstract

The purpose of this study was to characterize behavioral and physiological effects of a selective thromboxane (TP) receptor antagonist, SQ 29,548, in the C57Bl/6 mouse model. At 6 months of age, male mice were given either sham or drug i.p. injections for 3 days at a dose of 2 mg/kg each day. On the day after the final injection, mice were subjected to behavioral testing before brain collection. Left hemisphere hippocampi were collected from all mice for protein analysis via Western blot. Right brain hemispheres were fixed and embedded in gelatin and then serially sectioned. The sections were immunostained with anti-c-Fos antibodies. Prostaglandin analysis was performed from remaining homogenized brain samples, minus the hippocampi. Injection of SQ 29,548 decreased selective brain prostaglandin levels compared with sham controls. This correlated with robust increases in limbic-region c-Fos immunoreactivity in the SQ 29,548-injected mice. However, drug-treated mice demonstrated no significant changes in relevant hippocampal protein levels compared with sham treatments, as determined from Western blots. Surprisingly, injection of SQ 29,548 caused mixed changes in parameters of depression and anxiety-like behavior in the mice. In conclusion, the results indicate that administration of peripheral TP receptor antagonists alters brain levels of prostanoids and influences neuronal activity, with only minimal alterations of behavior. Whether the drug affects neurons directly or through a secondary pathway involving endothelium or other tissues remains unclear.

Keywords: anxiety; c-Fos; depression; thromboxane; thromboxane receptor.

Fig. 1

Prostaglandin analysis of mouse brain tissue. Six month old male mice were ip injected with DMSO vehicle (sham) or TP receptor antagonist, SQ 29,548 at 2 mg/kg for three days. Half brain hemispheres were collected for prostaglandin extraction and quantitation by LC-MS. Pro-inflammatory prostaglandins, PGD₂ and PGE₂, were decreased in drug treated animals compared to sham injected. TXB_2, was decreased in drug treated animals compared to shams. PGF2α and 6ketoPGF1α levels were not altered by drug treatment. Drug treatment attenuated overall prostaglandin levels, *p<0.05, n=5.

Fig. 2

Open field testing of TP receptor antagonist injected mice. Following three days of ip injection of DMSO vehicle (sham) or SQ 29,548 at 2 mg/kg, mice were examined by open field testing to quantify general locomotion behavior. Treatment with SQ 29,548 elicited decreases in clockwise body rotations and total body rotations as well as corner quadrant entries (Q1-4), *p<0.05, n=13.

Fig. 3

Elevated zero maze testing of TP receptor antagonist injected mice. Following three days of ip injection of DMSO vehicle (sham) or SQ 29,548 at 2mg/kg, mice were examined by elevated zero maze testing to quantify anxiety-associated behavior. Drug treatment did not significantly alter time spent in open arms compared to sham injected mice, n=13.

Fig. 4

Hanging tail suspension testing of TP receptor antagonist injected mice. Following three days of ip injection of DMSO vehicle (sham) or SQ 29,548 at 2 mg/kg, mice were examined by hanging tail suspension testing to quantify depression-associated behavior. Depression behavior was increased in SQ 29,548 treated mice compared to sham controls. Treatment with SQ 29,548 resulted in a decrease of both time spent mobile and total mobile episodes (start-stop events), *p<0.05, n=13.

Fig. 5

Forced swim testing of TP receptor antagonist injected mice. Following three days of ip injection of DMSO vehicle (sham) or SQ 29,548 at 2 mg/kg, mice were examined by hanging tail suspension testing to quantify depression-associated behavior. Drug treatment did not significantly change depression behavior measured by the forced swim test. Treatment with SQ 29,548 resulted in only an increase of counterclockwise body rotations, *p<0.05, n=13.

Fig. 6

Total brain PG levels significantly correlated with behavioral changes in TP receptor antagonist mice. Pearson correlation was used to measure the strength of the linear relationship between open field behavioral changes and total brain PG levels in individual mice injected three days. (A) A negative linear relationship between total prostaglandins and Q4 entries. (B) A negative linear relationship between total prostaglandins and open field max speed. (C) A positive linear relationship between total prostaglandins and tail suspension immobile episodes, n= 5.

Fig. 7

The TP receptor antagonist, SQ 29,548, selectively increased c-Fos immunoreactivity. Cerebral hemispheres collected from the sham and SQ 29,548 injected mice (n=13) were fixed and serially sectioned for immunohistochemical analysis. Serial brain sections from all mice were immunostained using anti-c-Fos antibody with Vector VIP as the chromogen. A) Regions of increased c-Fos immunoreactivity included a) Medial Orbital Cortex, b) Piriform Cortex, c) Posterior Hypothalamic Area, and d) Posterior Cortical Amygdala Nucleus. B) Numbers of immunopositive nuclei per region were counted and averaged +/-SD, **p<0.01, ***p<0.001. C) Representative anti-c-Fos staining is shown for each region.

Fig. 8

Pathways interconnecting elements of the limbic system. Schematic of limbic system neuronal interactions that include the increased c-Fos immunoreactive regions found in SQ 29,548 injected mouse brains.