Intrinsic neurons of fastigial nucleus mediate neurogenic neuroprotection against excitotoxic and ischemic neuronal injury in rat

Abstract

Electrical stimulation of the cerebellar fastigial nucleus (FN) elevates regional cerebral blood flow (rCBF) and arterial pressure (AP) and provides long-lasting protection against focal and global ischemic infarctions. We investigated which neuronal element in FN, perikarya or axons, mediates this central neurogenic neuroprotection and whether it also protects against excitotoxicity. In anesthetized rats, the FN was stimulated for 1 hr, and ibotenic acid (IBO) was microinjected unilaterally into the striatum. In unstimulated controls, the excitotoxic lesions averaged approximately 40 mm3. Stimulation of FN, but not dentate nucleus (DN), significantly reduced lesion volumes up to 80% when IBO was injected 15 min, 72 hr, or 10 d, but not 30 d, thereafter. In other rats, intrinsic neurons of FN or DN were destroyed by pretreatment with IBO. Five days later, the FN was stimulated, and 72 hr later, IBO was microinjected into the striatum. Lesions of FN, but not DN, abolished neuroprotection but not the elevations of rCBF and AP elicited from FN stimulation. Excitotoxic lesions of FN, but not DN, also abolished the 37% reduction in focal ischemic infarctions produced by middle cerebral artery occlusion. Excitation of intrinsic FN neurons provides long-lasting, substantial, and reversible protection of central neurons from excitotoxicity, as well as focal ischemia, whereas axons in the nucleus, probably collaterals of ramified brainstem neurons, mediate the elevations in rCBF, which do not contribute to neuroprotection. Long-lived protection against a range of injuries is an unrecognized function of FN neurons transmitted over pathways distinct from those regulating rCBF.

Fig. 1.

Effect of FN stimulation on excitotoxic lesions of rat striatum. A, Distribution of excitotoxic lesion in the right striatum 24 hr after microinjection of IBO (23 nmol in 360 nl of PB). Arrow indicates cannula track. Left striatum was injected with equal volume of PB. B, Reduction in excitotoxic lesion produced by 1 hr of electrical stimulation of FN 3 d before injecting IBO. C, Reduction in excitotoxic lesion produced by intraperitoneal pretreatment with MK801. Nissl-stained sections.

Fig. 2.

Effect of FN stimulation on area and distribution of unilateral excitotoxic striatal lesions in rat striatum. FN was simulated for 1 hr 3 d before injection of IBO. Lesion volumes were estimated 24 hr thereafter. A, Cross-sectional areas, expressed as mean ± SEM, at different levels of brain along the rostrocaudal axis at sites rostral to the interaural line. Each group represents five sham-stimulated (filled triangles), DN-stimulated (open circles), or FN-stimulated (open squares) rats. *p < 0.05. B, Distribution of lesions at different rostrocaudal levels in representative cases from each group described in A. Stippledepicts the extent of the lesion. Numbers represent the distance from interaural line. Note that only FN stimulation reduces lesion size.

Fig. 3.

Location of stimulation sites in the FN (filled circles) and DN (open circles) in 15 representative experiments each.CST, Cortical spinal tract; DN, dentate nucleus; FN, fastigial nucleus; STT, spinothalamic tract; VII, facial nerve nucleus;IV, ventricle IV.

Fig. 4.

Persistence of the neuroprotective actions of FN stimulation. Data are expressed as percentage of lesion volume in matched sham-stimulated rats (open bar). Eachsolid bar represents volume of lesions placed at various times between 1 hr of FN stimulation and microinjection of IBO.Hatched bar represents normalized data from all groups in which the DN was stimulated before IBO treatment. (n= 5–12; *p < 0.05). Mean volume for all sham stimulated rats was 40.9 ± 2.4 mm³(n = 24).

Fig. 5.

Changes in AP and rCBF in anesthetized rats elicited by electrical stimulation of FN with or without excitotoxic lesions of the nucleus. rCBF was measured by laser-Doppler flowmetry; IBO or vehicle were microinjected 5 d earlier. A, Intact rat during and at end of electrical stimulation of FN (50 Hz; 1 sec on, 1 sec off stimulus current five times threshold). Note rapid rise in rCBF and AP, which recovers to baseline after 1 hr, characteristics of the fastigial pressor response. B, FN-lesioned rat. Note preservation of stimulation-evoked elevations in rCBF and AP, despite destruction of intrinsic neurons of FN as verified histologically.

Fig. 6.

Excitotoxic lesions of FN reduces the salvage produced by FN stimulation of both excitotoxic lesions and focal ischemic infarctions. Intrinsic FN neurons were destroyed by IBO 5 d before FN stimulation or sham stimulation. Average cross-sectional area (n = 5–6 per group; mean ± SEM; *p < 0.05, FN-stimulated compared with sham-stimulated) are plotted relative to distance from interaural line as in Figure 2. Groups of rats comprising sham-stimulated (filled triangles), FN-stimulated without excitotoxic lesions (open circles), and FN-stimulated (open squares) with excitotoxic lesions of FN were compared. A, Effects on excitotoxic lesions. IBO was microinjected into striatum 3 d after stimulating the FN, and rats were killed 24 hr thereafter. B, Volume of focal ischemic infarctions. Note that destruction of intrinsic lesions of FN abolish neuroprotection with either lesion.

Fig. 7.

Representative lesions of FN produced by injection of IBO into FN 9 d earlier visualized at low (scale bar, 1.5 mm) and higher (scale bar, 0.125 mm) magnifications. A,B, Naive control. Arrow indicates left FN. Note large Nissl-stained FN neurons at higher power.C, D, IBO treatment. The broken lines outline the area of complete destruction of local neurons with reactive gliosis 9 d after microinjection of IBO. Note loss of large neurons. E, F, The FN 9 d after injection of vehicle. Note accumulation of small stained cells about some neurons.

Fig. 8.

Effect of FN stimulation in spontaneously hypertensive rats with or without excitotoxic lesions of FN on focal ischemic infarctions produced by occlusion of MCA. A, Naive rat. B, Twenty-four hours after MCA occlusion.C, MCA occlusion immediately after 1 hr of FN stimulation. The rat was killed 24 hr later. Note reduction of lesion area and distribution. D, MCA occlusion immediately after 1 hr of FN stimulation in a rat in which the FN was destroyed by IBO 5 d earlier. Note that the stimulation no longer salvaged the lesion compared with C. Nissl staining.

Fig. 9.

Possible neuronal circuits mediating neuroprotection and changes in rCBF, rCGU, and AP elicited by stimulation of the FN. A, Neuroprotection results from excitation of intrinsic neurons of FN, which relay through unidentified pathways to protect cortex and striatum. The reduction in AP, rCBF, and rCGU evoked by chemically stimulating FN depends on RVL because bilateral lesions block these responses (Chida et al., 1990).B, Elevations in rCBF and AP elicited by electrical stimulation of FN result from antidromic excitation of brainstem neurons projecting to FN and, as proposed (Chida et al., 1990), collaterally to RVL. It is excitation of reticulospinal neurons of RVL which initiate the elevations of AP and rCBF (for review, see Reis et al., 1994). SG, Sympathetic ganglion.