The results of this preliminary experiment provide evidence that HCRT neurons can be grafted into the pons and survive in an area where such neurons are normally not present. Very few HCRT-ir neurons survived until day 36, but those that did survive had clearly established processes and appeared in size and morphology like normal adult HCRT-ir neurons. A number of experiments have shown that a high percentage (95%–99%) of transplanted cells die after grafting.21,22
Our findings are consistent with most graft studies of other types. Cell death after transplant occurs via apoptosis or necrosis.23–25
Apoptotic death is distinguished from necrosis by morphologic and biochemical changes, including preservation of membrane integrity and diminution of cellular volume, both present in apoptosis.26
Since, as seen in , on day 6 the size of the somata of grafted HCRT-ir neurons was dramatically reduced and the membrane was conserved, we suggest that an apoptotic cell death was triggered, probably as a consequence of loss of trophic factors.
The survival of grafted neurons is influenced by many factors. Perhaps, the most important is the age of the donor, since it has been shown that young embryonic tissue survives better than neonatal or adult donor tissue.27
Here, we used tissue from rat pups that were 8 to 10 days old. We did not take tissue from rat fetuses, since the HCRT neurons have been described to fully develop by postnatal day 10.28
Future studies may consider using fetal rat tissue so that the undifferentiated neurons may be placed in a site where, with appropriate stimuli, HCRT expression may occur.
Data from transplant studies indicate that the survival of cells and their ability to establish connections also depend on release of diffusible factors such as hormones, neurotransmitters, or neurotrophic factors.29,30
We assume that the pons is able to secrete factors that entice HCRT axonal growth because normally this region is heavily innervated by HCRT fibers.8
These factors are still present and promote cell growth in adults, since a few HCRT neurons were evident 3 days after transplant. Addition of neurotrophins or caspase inhibitors in the transplant media and/or inhibition of glia could improve survival.29,30
It is also known that to prevent rejection, hosts are immunosuppressed through injections of cyclosporin.31
HCRT-containing neurons project to the entire brain and spinal cord, providing especially heavy innervation to forebrain and brainstem neuronal populations implicated in wakefulness.8
In our study, we placed the grafts in the pons, since extensive data indicate that this region is involved in regulating sleep-wakefulness9
and receives HCRT innervations that modulate sleep.10
Thus, even though narcolepsy is characterized by loss of HCRT neurons in the lateral hypothalamus,2,3
it is necessary to place the grafts of HCRT neurons in target regions responsible for the behavior, which has been done with Parkinson disease.32
Once the problem of survivability of the HCRT neurons is improved, it will be necessary to determine impact of the transplanted HCRT neurons on sleep-wake behavior. In the present study, we did not examine such behavior because the parameters influencing HCRT survival are still not fully understood. It will be necessary to measure HCRT release, most likely in the CSF. We recently demonstrated that CSF HCRT levels decline with HCRT neuronal loss and that there is no subsequent recovery in HCRT levels once the HCRT neurons are destroyed.7
This is a particularly useful finding for transplant studies, since we can expect an increase in HCRT levels with HCRT transplants. However, it may be necessary to wait several months before appreciable increases in CSF levels of HCRT and/or a change in sleep behavior is evident in transplanted animals. For instance, some suprachiasmatic nucleus-lesioned animals bearing suprachiasmatic nucleus grafts remain arrhythmic for several months before animals begin to exhibit circadian rhythms of locomotor activity.15,33–35
This delay in emergence of behavior may reflect the time required for appropriate reestablishment of neuronal connectivity between the donor and the host.
The lateral hypothalamus where the HCRT neurons are located contains a mixed variety of neuronal phenotypes,36
and the HCRT neurons represent a small proportion relative to other phenotypes such as the melanin concentrating hormone-containing neurons. Clearly, in our method, other phenotypes, including the HCRT neurons, were transplanted. It would be preferable to transplant only HCRT neurons, perhaps using a transgenic mouse model where the green fluorescent protein identifies HCRT neurons. In these mice, the lateral hypothalamus could be dissected and the green fluorescent protein-HCRT neurons separated under a microscope and implanted into host animals. This method would also provide a way of quantifying the number of HCRT neurons that are being transplanted. The host animals would be mice where the HCRT neurons have been eliminated37
or mice that lack the HCRT gene,38
since they represent excellent models for testing the efficacy of transplant or gene therapy. In these models, the symptoms of narcolepsy are present, and targeted transplants could be used to reverse 1 or more of the symptoms. In the mouse model, downstream effector neurons are still responsive to HCRT, since ectopic HCRT expression and HCRT administration reverse the behavior.39
One has to be cautious, since neuronal transplant and gene therapy have limitations40,41
and might not be suited to treat a disease such as narcolepsy, which can be managed by pharmacotherapy. However, pharmacotherapy also has its limitations, such as tolerance and side effects, and thus the need to develop alternative strategies.