Neuroseienee Letters, 143 (1992) 151 154 t3 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00
Responses of regional cerebral blood flow to intravenous administration of thyrotropin releasing hormone in aged rats O. I n a n a m i , K. Ohno and A. Sato Department o["Autonomic Nervous System, Tokyo Metropolitan Institute q/" Gerontology. Tokyo ~Japan ) (Received 22 May 1992; Accepted 27 May 1992)
Key words: Thyrotropin releasing hormone: Regional cerebral blood flow; [~4C]Iodoantipyrine: Vasodilatation: Aging: Rat The effects of i.v. administration of thyrotropin releasing hormone (TRH) on regional cerebral blood flow (rCBF) were examined in both healthy adult (3 5 months old) and healthy aged (24 25 months old) male Wistar rats under halothane anesthesia. The rCBFs in 9 different brain regions cerebral cortex, caudate putamen, hippocampus, thalamus+hypothalamus, superior colliculus, inferior colliculus, cerebellum, pons, and medulla were measured by [~4C]iodoantipyrine method. In the adult rats, i.v. administration of TRH (300/ag/kg) produced significant increases in rCBFs in cerebral cortex, caudate putamen, hippocampus, thalamus+hypothalamus and superior colliculus. In the aged rats, the rCBFs in all brain regions measured did not change significantly by TRH administration. From these results, it is suggested that the system involved in TRH-induced vasodilatation of cerebral blood vessels was impaired with aging.
Thyrotropin releasing hormone (TRH) regulates the release of thyrotropic hormone (TSH) from the pituitary gland. In addition, TRH has other effects on central nervous function. For example, TRH improves mental depression , and in patients with Alzheimer's disease, acute treatment with T R H produces a significant increase in arousal and also an improvement in mood, as well as a modest improvement in semantic memory . T R H shortens pentobarbital-induced sleeping time in rodents [4, 8]. It is noteworthy that cortical cerebral blood flow (CBF) increases following either an i.v. or i.c.v, administration of TRH in anesthetized animals [7, 9, t0, 12]. It has been suggested that the TRH-induced increase of regional cerebral blood flow (rCBF) is not secondary to the TRH-induced increase in systemic arterial blood pressure [7, 12] or to the increased metabolism in the brain , but to activation of intracerebral intrinsic vasodilative nerve fibers [10, 11]. In atropinized animals, the increase in cortical CBF following T R H administration is much attenuated, suggesting that a cholinergic vasodilative system contributes to the TRH-induced increase in cortical CBF . An i.v. or s.c. administration of TRH increases both the activity of neurons in the nucleus basalis of Meynert (NBM) which project to the cortex  Correspondence: A. Sato, Department of Autonomic Nervous System, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho, Itabashiku, Tokyo 173, Japan.
and the extracellular concentration of ACh in the cerebral cortex [5, 23]. The consistent nature of these results lead us to propose that the TRH-induced activation of the cholinergic fibers originating in the NBM is instrumental in producing the TRH-induced increases in the cortical CBF. It has been reported that in the human, aging is associated with a dysfunction of the central nervous system, and in particular a reduction of the activity of choline acetyltransferase which is involved in the synthesis of acetylcholine in the cerebral cortex and hippocampus . This evidence has prompted the question whether the TRH-induced increase in rCBF alters during aging. Therefore, the present experiment was performed to compare the TRH-induced increase in rCBF in various brain regions between healthy adult and aged Wistar rats. The experiment was performed using 12 healthy adult (3 5 months old, 280-345 g) and 12 healthy aged (24-25 months old, 390450 g) male Wistar anesthetized rats. The 12 adult and 12 aged rats were divided into the following 4 groups, a saline-injected adult group (1), a TRH-injected adult group (2), a saline-injected aged group (3) and a TRH-injected aged group (4). Each group consisted of 6 rats. During surgical procedures, halothane concentration for anesthesia was maintained at 1.2 1.5% in a gas of 100% oxygen through a tracheal catheter. Respiration
[ ] saline Ir'AITRH (300jug/kg)
Fig. 1. The effects of TRH administration (300 pg/kg, i.v.) on the rCBF in 1.0% halothane anesthetized adult rats (A) and aged rats (B). White and hatched columns show rCBFs in the saline-injected group (n=6) and the TRH-injected group (n=6), respectively. Each column and vertical bar represents the mean + S.E.M. Cx, cerebral cortex: CPu, caudate putamcn: Hpc, hippocarnpus; Thai, thalamus + hypothalamus; Sup.C, superior colliculus; lnf.C, inferior colliculus: Crb, cerebellum. "P < 0.05, **P < 0.01. significant difference from the saline-injected adult group by Student's t-test.
was maintained by an artificial respirator (Harvard pump 681, USA). The right femoral vein and artery were catheterized. After completing the surgical procedures, halothane concentration was reduced to 1.0% in the inspiratory gas which was readjusted to contain a mixed gas of 22-25% oxygen in nitrogen. The end-tidal CO2 and 02 were kept at around 4.0% and 18.0%, respectively, by adjusting the rate of the respirator. Rectal temperature was maintained at 37.0-38.0°C by means of a lamp and a heating pad. After all surgical procedures had been completed, at least 60 min elapsed before measuring the rCBF. At the beginning of the experiments, a sample of arterial blood (0.2 ml) was also collected via the right femoral arterial catheter for determination of hematocrit (Ht), paO2 and p,C02 (170 pH/Blood Gas Analyzer, Corning, USA). In the experimental TRH-injected adult and aged groups, a dose of 300 pg/kg TRH (g-pyroglutamyl-L-histidyl-u-prolinamide u-tartrate monohydrate; Nippon Shinyaku, Kyoto) dissolved in 200 pl of saline was administered i.v. A previous report from our laboratory demonstrated that cortical CBF was increased with i.v. administration of 300 pg/kg TRH in adult rats using laser Doppler flowmetry . In the control experiment,
200 pl of saline was administered to adult and aged rats (groups 1 and 3). Five min after injection of either TRH or saline, the rCBF was measured according to the Kety's principle, using [14C]iodoantipyrine ([t4C]IAP, spec. act. 1.85 GBq/ mmol, Amersham, UK). This method has been previously described . Sixty seconds after the start of [14C]IAP infusion, the animal was sacrificed by decapitation. Concentrations of [~4C]IAP in the 9 different bilateral brain regions (i.e. cerebral cortex, caudate putamen, hippocampus, thalamus+hypothalamus, superior colliculus, inferior cotliculus, cerebellum, ports, and medulla) were obtained by tissue dissection as per the technique employed by Ohno et al. . All the comparisons of rCBF were analyzed statistically by Student's t-test. The hematocrit, PaO2 and paCO2 in these 4 groups are shown in Table I. The paO2 in the aged groups were significantly lower than those of the adult groups. However, these values in the aged groups were within the normal range . Fig. IA summarizes rCBF in all 9 different brain regions in the saline-injected adult group (white column, n=6) and in the TRH-injected adult group (hatched column, n=6). In the TRH-injected group the rCBFs in cer-
ebral cortex, caudate putamen, hippocampus, thalamus+hypothalamus and superior colliculus were significantly increased compared to the saline-injected group. The rCBFs in cerebral cortex, caudate putamen, hippocampus, thalamus+hypothalamus and superior colliculus in the saline-injected group were 102 + 9, 89 +_ 8, 72 +_ 6, 85 + 7 and 89 _+ 9 (means + S.E.M.) ml/ 100 g/ min, respectively, whereas the rCBFs in the corresponding regions in the TRH-injected group were 176 _+ 17, 173 + 16, 108 + 11,130 + 15and 145 + 20ml/lOOg/min, respectively. The rCBFs in the other brain regions were not significantly different between these 2 groups. These results showed that T R H administration produced significant increases in rCBFs in 5 of the 9 brain regions measured. These results are consistent with those reported previously [9, 12]. Fig. 1B summarizes rCBFs in all 9 different brain regions in the saline-injected aged group (white column, n=6) and in the TRH-injected aged group (hatched column, n=6). The rCBFs in the saline-injected aged group (white column in Fig. 1B) were not significantly different from those in corresponding regions in the saline-injected adult group (white column in Fig. IA). It was shown that the rCBFs under resting condition were well maintained during aging. In aged rats there were no significant differences in rCBFs between the saline-injected group and the TRH-injected group in any brain regions tested (white column and hatched column in Fig. 1B), suggesting that TRH-administration produced no increases in rCBFs in the aged rats. Ohata et al.  reported that the rCBFs in the frontal and occipital cortex, caudate putamen, hippocampus, superior colliculus and cerebellum were independent of aging but those in thalamus+hypothalamus, inferior colliculus, pons and medulla are significantly decreased in aged (24 months old), conscious Fischer-344 rats in comparison with young adult (3 months old) rats and middleaged (12 months old) rats. In our study, the absolute rCBFs in all 9 brain regions measured were not decreased by aging. This discrepancy may be due to difference of strain (Fischer-344 and Wistar) or anesthetic condition (conscious or halothane anesthesia). In this experiment, the increase of rCBFs in the cerebral cortex, caudate putamen, hippocampus, thalamus+hypothalamus and superior colliculus as a result of T R H administration were almost abolished with aging. T R H injected i.v. penetrates to the brain through blood brain barrier  and acts on T R H receptor in the brain and may activate an intrinsic vasodilative pathway . Although the exact mechanism of vasodilative action in the brain of T R H is unknown, several proposals exist. We suggest that intracranial cholinergic vasodilative system originated in NBM contributes to the TRH-induced
increase in the cortical CBF. This suggestion is based on the facts that the TRH-induced vasodilative response was attenuated by i.v. injection of atropine (a muscarinic cholinergic receptor antagonist)  and that TRH-administration produced the increase of neuronal activities of NBM and consequent release of ACh in the cerebral cortex . It was also demonstrated that the electrical stimulation of NBM produced increases in cortical CBF and a release of ACh in the cortex [3, 14]. On the other hand, Koskinen  suggested from the study with transection of the brainstem that the increase in rCBF caused by T R H was probably mediated at the submesencephalic level. Furthermore, there are reports that prostaglandin metabolism and/or ~-receptors are involved in cerebral vasodilatation induced by i.v. administration of T R H [6, 9]. The results of this study indicate that some function of the TRH-induced vasodilative pathway en route from the blood brain barrier to blood vessels in the brain deteriorate with aging. The function of the blood brain barrier is well maintained with aging . The increase of CBF and ACh release in the parietal cortex to local electrical stimulation of the NBM is also well maintained in the aged rats . However the effect of aging upon the other vasodilative systems have not been reported. In contrast, the concentration of T R H receptors in the brain was decreased m aged rats . Therefore, it is possible that the present attenuation of TRH-induced vasodilatation of cerebral blood vessel with aging is due to the decrease of the function of T R H receptors and/or the impairment of the function of TRH-related vasodilative systems other
TABLE I H E M A T O C R I T (Ht), A N D BLOOD GASES (17~,0~ A N D p,,CO3) OF (1} S A L I N E - I N J E C T E D A D U L T G R O U P (n-6), (2) T R H - I N J E C T E D A D U L T G R O U P 01-6), (3) S A L I N E - I N J E C T E D A G E D G R O U P (n=6l A N D (4) T R H - I N J E C T E D A G E D G R O U P (n 6) Values of hematocrit, p,,O2 and p:,CO 2 are represented by mean ± S.E.M. p,O~ and p~,CO3 were measured frorn a sample of arterial blood which was collected before administration of TR H or saline. Adult groups Salineinjected (1) Ht (%)
45.3 + 0.6
45.7 + 0.6
102 + 2
104 +_ 5
31 + 1
32 + I
Aged groups Salinerejected 13}
45.0 + 2.3
44.5 + 1.7
88 + 1"*
89 + I'*
32 + 2
P,,02 (mmHg) p,CO~ (mmHg)
"*P < 0.01, significant difference from the saline-injected adult group by Student's t-test.
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