Modifications of the distribution of substance P-like immunoreactivity in the hipotalamus of the cat after electroacupuncture

Dr. J. C. Crespo, Dr. M. Munoz, Dr. J. Vazquez, Dr. E. Morales

Summary: The distribution of Substance P in the fibrillar structures of hipotalamus of the cat brain, and its modification with low frequency’ electroacupuncture (EA). The frequency was 3-5 Hz and the intensity 8 mA. Periferic stimulation with EA was applied in Hegu and Kumlun points, for 20 min, have been studied using the indirect immunocytochermstry technique. An increase in the soma immunoreactivily of Substance P (Appearance of some inmunoreactives) after stimulation with EA has been observed in the posterior hipotalamus, nucleus ventro medialis hipotalamus, periventicular region y perifornix IR-SP soma fibres have also been seen in some areas of the internal capsule. These observations place the said IR-SP soma modifications in the hipotalamus of the cat brain when subjected to low frequency EA.

Key words: Substance P, Electroacupuncture, Hipotalamus, Immunocyt.ochemistry. Cat


Electroacupuncture (EA) diminishes pain perception involving the met-enkephalin system (Vacca-Galloway et. al., 19&127;3; Munoz, 1986; Vazquez et al., 1990). It has been observed that EA incluces alterations in the distribution of met-enkephalin, like immunoreactivity in the cat thalamus (Vazquez et al; 1990). Substance P (SP) is a neuropeptide probably related to nociceptive transmission (Lembeck, 1983; Luque, 1988) and thus, with the met-enkephalin system (Hughes 1975); Holdfelt et al.; 1975; Jessel and Iversen, 1977). SP is localized in the small diameter fibres of the posterior horn of the spinal cord, which are considered to be involved in nociceptive transmission. Moreover, alteration of SP-immunoreactivity in the spinal cord after EA has been demonstrated (Vacca Galloway et. al., 1985).

The cerebral basal nucleus shows SP-inununoreactive fibres in the striatonigral pathway (Glowinsky et. al.; 1982), the pallidus-caudate system (Floret and Martinez-Lage, 1983) and the amygdaloid complex (Bouras et al., 1986). In addition, it has been described that the injuries in the caudate nucleus abolish EA analgesia (Gonzalo, 1979), Takeshige et al. 1979).

Thus, in order to obtain new data on nociceptive transmission, the influence of EA or SP-immunoreactivitv on the cerebral hipotalamusi have been investigated.

Materials and methods

Experimental procedure

Six adult cats (2-3 Kg) were divided into two groups: contra group (three cats) and experimental group (three cats). Animals were anaesthetised with ketamine (50 mg/kg body weight) (Conrath et al., 1983); then. periferic stimulation with EA if low frequency was applied in Hegu and kumlun points, for 20 min. The correct stimulation was confirmed by the rhythmic contractions observed.

Tissue processing

Animals of both groups were perfuse first with buffer (&127;99 ml), and then, with 4% paraformaldehyde diluted in Sorensern buffer (Paese, 1962). The encephalon was then postfixed in the same fixative for 12 h and afterwards rinsed in several baths of sacarose in Sorensen butler. After washing the encephalon was frozen in liquid nitrogen and 80 um frontal sections were obtained in a cryostat.

Immunocytochemical staining

For the immunocvtochemical detection of SP indirect techniques were used (Nakane-and Pierce, 1996, Falini and Taylor, 1983, Conrath et al. 1986). Tissue sections were immersed in 0,3 %. H202 in methanol to eliminate endogenous peroxidase, then after hydration, sections were incubated with 1% normal sheep serum in 0, 3 % triton X-100. Sections were then incubated overnight with rabbit anti-SP antibody (1:1000, Cambridge Research Biochemicals, Cambridge, UK) As secondary layer sheep anti-rabbit IgG horseradish peroxidase-conjugated antibodies were used at a dilution 1:200. Peroxidase was visualized with 3.3′ diamenobenzidine. The following controls were used: a) preabsortion of the first antibody with SP; b) omission of the different antibodies.

The mapping was carried out according to the stereotaxic atlas of Jasper and Ajmone-Marsan ( 1996).