Transplantation of hypocretin neurons into the pontine reticular formation: preliminary results

Abstract

Study objectives: The sleep disorder narcolepsy is now considered a neurodegenerative disease because there is a massive loss of neurons containing the neuropeptide, hypocretin, and because narcoleptic patients have very low cerebrospinal fluid levels of hypocretin. Transplants of various cell types have been used to induce recovery in a variety of neurodegenerative animal models. In models such as Parkinson disease, cell survival has been shown to be small but satisfactory. Currently, there are no data indicating whether hypocretin neurons can survive when grafted into host tissue. Here we examined the survival of hypocretin-containing neurons grafted into the pontine reticular formation, a region traditionally regarded to be key for rapid eye movement sleep generation.

Design: In 2 experiments, a suspension of cells from the posterior hypothalamus of 8- to 10-day old rat pups was injected into the pons (midline, at the level of the locus coeruleus) of adult rats. Control rats received cells from the cerebellum, tissue that is devoid of hypocretin neurons. In the first experiment (n = 33), the adult rats were sacrificed 1, 3, 6, 12, 24, or 36 days after transplant, and cryostat-cut coronal sections of the brainstem were examined for presence of hypocretin-immunoreactive neurons. In the second experiment (n = 9), the transplant medium was modified to include agents that stimulate cell growth, and recipient rats were sacrificed 9, 12, and 36 days after receiving the graft.

Settings: Basic neuroscience research laboratory.

Measurements and results: In the first experiment, clearly defined hypocretin-immunoreactive containing somata and varicosities were visible in pons of rats sacrificed 1 day after grafting of posterior hypothalamic cells but not in rats receiving cerebellum tissue. The hypocretin-immunoreactive somata were not visible in rats sacrificed at 12, 24, or 36 days, indicating that the neurons had died. However, in the second experiment, where enriched transplant medium was used, clearly defined hypocretin-immunoreactive somata with processes and varicosities were present in the graft zone 36 days after implant. These somata were similar in size and appearance to adult rat hypocretin-immunoreactive neurons.

Conclusions: These results indicate that hypocretin neurons obtained from rat pups can be grafted into a host brain, and efforts should be made to increase survival of these neurons.

Figure 1

Hypocretin-immunoreactive (HCRT-ir) neurons in Experiment 1. Representative HCRT-ir neurons in rat pups are shown in panel A. A cell suspension containing HCRT-ir neurons was placed in the pons of adult rats and examined 1 day (panel B) or 6 days (panel C) after grafting. HCRT-ir neurons were not evident when cerebellum tissue was grafted into the pons (panel D). The HCRT-ir neurons present 1 day after grafting were round and smaller than in the donor rat pups (panel A versus panel B or C).

Figure 2

Hypocretin-immunoreactive (HCRT-ir) neurons in host rats sacrificed 9 (photo A and B) or 12 days (photo C) after grafting of posterior hypothalamic tissue into the pons (Experiment 2). In A and B, double arrows identify HCRT-ir neurons that are round in rats sacrificed 9 days after grafting. In A, the arrowhead identifies a neuron with a disintegrated membrane. On day 12, HCRT-ir neurons with an enlarged nucleus and thin cytoplasm are evident, suggesting that these neurons might be dying.

Figure 3

Hypocretin-immunoreactive (HCRT-ir) neurons with processes in adult rats sacrificed 36 days after receiving a graft in the pons of dissociated posterior hypothalamic tissue from 8- to 10-day-old rats pups (Experiment 2). Photos taken from host rats show that some of the grafted tissue accumulated in the fourth ventricle (IV) and HCRT-ir neurons with a morphology and size found in adult rats were evident. Inset B represents a close-up of the section identified by a box in A, and 2 neurons (1 out of the focal plane) with processes are evident. In photo C, 3 HCRT-ir neurons are evident in the graft zone, and 1 is present in the periaqueductal grey (PAG) area. All of the HCRT-ir neurons have clear processes with varicosities (identified by arrows in B and C). The arrow in photo A points to the graft in the ventricle.