NEUROGENESIS

HISTORY
Can neurons regenerate? Are the brain and spinal cord nerve cells able to repair themselves or replicate? This is a topic that has received more attention in the past decade than it enjoyed in the previous 50 years. To appreciate this some historical review is in order.

 But first, here’s a story to begin this section. Recently, in 2006, Dr. Michael Fehlings extracted adult stem cells, not embryonic fetal stem cells, from adult rats and injected them into the spinal cords of rats that had had their spinal cords crushed. He found that when he treated these “implanted” rats with a cocktail of human growth hormone, and minocycline, these rats were able to make new myelin sheaths around the nerve fibers which restablished the ability of the nerves to conduct their signals from the brain to the muscles of the limbs. The rats were able to again bear weight on their legs (and arms) and to walk with better coordination. The implanted adult stem cells had become new perineural cells which made and repaired the myelin. We didn’t think this was possible. 

Now, with this in mind and remembering the “cocktail” approach of my stroke protocol discussed in that section of this website, read on. If we focus on fixing the patient rather than on our own arrogance, or on looking for the “one drug” that will cure the patient and make us millions of dollars, then we’ll see progress.  Now let’s go back in time.

In the early 1900’s Dr. Santiago Ramon y Cajal postulated that mammals are born with a given number of neurons. These neurons, barring any direct insult such as trauma, ischemia, or poisoning, are static in number and remain functional throughout the life of the organism. This view was generally accepted  and for the most part uncontested for the subsequent 70 years. 

 Dr. Joseph Altman, using a relatively new technology in the 1960’s, injected animals with radioactive thymidine and looked for its presence in neurons. Its presence would denote the formation of new DNA sequences and therefore, by extension, new cells. And indeed he found it. However, as is often the case with such discoveries that call into question basic tenets of science and utilize novel methodologies, his findings were largely ignored.

 The concept of the static brain gathered momentum with the research of Dr. Pasko Rakic at Yale. In the 1980’s he used much the same technique used by Dr. Altman at MIT, radioactive thymidine labeling of new neurons in primates. However, unlike Dr. Altman, he was unable to identify any uptake of thymidine in his primate model. This result appears to have confirmed Dr. Cajal’s findings more than a half century before and scientists were satisfied that in the adult mammal no neurogenesis occurs.

 With the advent of electron microscopy, however, new technology permitted a look at neurons from a much closer perspective. Dr. Michael Kaplan presented electron micrographs in the early 1990’s that seemed to demonstrate nascent neurons throughout the brain in adult animals. Unfortunately, this data again met with a generalized disinterest and failed to change the predominant scientific views of Cajal.

 Since the mid 1990’s, however, more and more data from well designed studies has begun to accumulate. Dr. Ronald Duman has demonstrated the propagation of trophic and growth factors (brain derived growth factor or BDGF) which result from brain tissue exposure to antidepressants such as fluoxetine and other SSRI antidepressants. These trophic factors stimulate neurogenesis by 50% in the hippocampus of the rat. Dr. Duman believes this observation may account for the 7-14 day delay in the clinical benefits of antidepressant medications and that the effects of the medications are dependent on the generation of new neurons rather than exclusively on the inhibition of the reuptake of serotonin. He has also demonstrated that stress decreases these same trophic factors which are stimulated by exposure to fluoxetine.

 Dr. Elizabeth Gould has data in primates that shows when they are exposed to novel environments, the hippocampal neuron count increases dramatically, but under stressful conditions for prolonged periods of time, the count decreases. Additionally, in the novel environment, the dendritic number and density increases. This may account for the different findings of Rakic in whose studies the primates were not constantly exposed to novel environmental stimuli.

 To add even more support to the concept of neurogenesis in the adult mammalian brain, Dr. Jonas Frisen published a paper in 1999 identifying stem cells in the brain. These adult stem cells are stimulated to differentiate into neurons by a cascade of proteins and trophic factors. Dr. Frisen is working to identify drugs that might trigger this cascade and subsequent differentiation of adult stem cells, specifically in the substantia nigra of a rat model of Parkinson’s disease. He has data to show a reversal of limb paralysis after 5 weeks of treatment with one of his drugs suggesting a restoration of dopamine neurons in the brains of these animals.

So by way of review, since 1995 the field of neuroscience has progressed from a belief that was spawned at the turn of 1900 by Dr Cajal’s papers postulating the absence of neurogenesis in the adult mammalian brain to the demonstration of the presence of adult stem cells in the mammalian brain which are capable of differentiating into new neurons as a result of stimulation of the trophic protein cascade by certain drugs and chemicals and that then can be integrated into the brain’s functional network. 

 And why wouldn’t this be the case? After all, mammals have stem cells in every tissue. Erythropoesis is probably the best known example of adult stem cell differentiation and it is stimulated by erythropoietin, a hormone, and by L-carnitine, and by taurine, and by somatotropin or human growth hormone, to name only a few. Dermis and endothelial tissues regenerate similarly and can be stimulated to do so by specific hormones and chemicals such as basic fibroblast growth factor or BFGF that appear to drive the cascade of trophic proteins responsible for this differentiation. 

So let’s look at some of the most current data that demonstrate neurogenesis which occurs as a result of adult stem cell activation in response to specific chemicals and hormones. I have already discussed some of these substances in previous chapters with regard to their neuroprotective attributes and it is remarkable that these same biochemicals can also induce neurogenesis. As one reads the following review, and taking into consideration the information presented in the previous chapters, one has to wonder if this were any era other than this one of burdensome litigation and overbearing restrictions by regulatory agencies and ignorance and arrogance of the unscientific clinician and his and her parent organizations, would we not have implemented protocols for both emergent and chronic management of brain injury and stroke that could have saved suffering?. If the results of Drs. Altman and Kaplan had not been ignored in the 1960’s and 1980’s, would Parkinson’s disease be only a historical reference in 2007 because of research such as that being done by Jonas Frisen? 

HUMAN GROWTH HORMONE
With this in mind, let’s start with somatotropin or human growth hormone and its production of IGF1 are both now well established as stimuli for neurogenesis in the neuroscience literature.. It’s not surprising that in the developing mammal, growth hormone increases IGF binding protein-3 and thereby promotes proliferation of both neuro and gliogenesis (Ajo, et.al, 2003).  However, of much greater significance is the effect of somatotropin and IGF1 on adult neural stem cells. As recently as the year 2000 Aberg et. al. published one of the first studies to demonstrate the effects of IGF1 on adult neural stem cells. Peripheral infusion of IGF1 dramatically increased the number of these stem cells and selectively induced neurogenesis of their progeny. The results were interpreted with cautious enthusiasm at the time. In the past 8 years, however, numerous studies have substantiated and expanded upon these results. Dr. R.J. Lichtenwalner’s group at Wake Forest has been studying the effects of human growth hormone and IGF1 on adult neural stem cells in the hippocampus. Based on the observations that IGF1 declines with age and correlates with the senescent cognitive decline, they hypothesize a causal relationship. In 2001 they published a study in the Neuroscience that demonstrated that not only was the rate of newly generated cells age-related, but there was a 60 per cent reduction in the differentiation of these newly generated cells into neurons, but restoring IGF1 levels in these animals resulted in a 3 fold increase in neuron production. These results were expanded in a 2006 paper in Journal of Neuroscience Research showing in growth hormone deficient rats, that although the rate at which new cells were generated did not appear to be affected by the growth hormone deficiency, their survival as functional neurons is dramatically impaired. 

 This antiapoptotic effect of  human growth hormone has support from a number of studies. Dr. Torres-Aleman (2000, 2003) found a neuroprotective role for IGF1 and its production of neurotrophic factors, calling it a “neuroprotective surveillance”. Perez-Martin, et.al. report similar results in the bovine brain tissue (2003).  Drs. Sun and Bartke (2005, 2007) found that growth hormone and IGF1 levels are maintained at levels significantly higher in the long-lived Ames dwarf mice compared with normal strains of mice. Despite their advanced age, neurogenesis and the mRNA levels in the brains of these mice and their cognitive function remain at levels consistent with young mice of normal strains and do not decline as do those levels in the brains of normal mouse strains. They believe this to be due to the growth hormone and IGF1 activation of increased phosphorylation of Akt and the subsequent cyclic AMP responsive element-binding protein cascade maintaining neuronal structural integrity. Their proposed mechanism of action of IGF1 on these protein cascades is supported by Ortaegi, et.al. (2006). Interestingly, they did not note a difference in number of new cells, but in the number of new neurons as compared to aged normal mice strains, again supporting a role of growth hormone and IGF1 in preventing the apoptosis and in the maintenance of newly generated cells allowing them to progress in differentiation to functional neurons.

 The Swedish group of Anderson, Aberg, Nilsson, and Ericksson has also presented a number of studies demonstrating growth hormone and IGF1 stimulating neurogenesis in the adult mammalian brain (2000, 2002). They have also found that exercise similarly stimulates neurogenesis and that growth hormone release during exercise mediates this effect. Dr.Torres-Aleman’s group (2001) has also demonstrated the exercise both prevents and reverses cognitive and functional decline in aged rats of specific cerebellar neurodegenerative strains and in normal strains with experimentally-induced lesions in a variety of brain regions. The independent variables were the animals’ performance in spatial memory tasks as well as motor coordination tasks. They also noted that administration of an anti-IGF1 antibody blocks the exercise-induced neuroprotection.

 Dr. Lichtenwalner has continued to evaluate neurogenesis in ischemic brain tissue and has an excellent review of the current state of the research as of 2006 (Lichtenwalner and Parent, 2006). In this paper they discuss what we have already been presenting, that not only is the mammalian brain capable of forming new neurons from progenitor neural stem cells and then integrating them into existing neuronal networks, but that this process is augmented following certain brain injuries, both traumatic and ischemic. Following such injury, the progenitor cells migrate into the injured area and form neurons “in otherwise dormant forebrain regions.” 

 Let’s look at this more closely. We have already discussed the contribution of nitric oxide and its potential for minimizing the effects of brain ischemia through its ability to increase blood flow via its vasodilatory effects and its ability to block the neurotoxic secondary damage of excitatory neurotransmitters aspartate and glutamate which are released during and after ischemic injury. A group of 3 Dr. Zhangs with Wang, Lu, Lapoint and Chopp published a report in Neurology in 2001 showing that a nitric oxide donor compound administered to rats following an experimentally induced middle cerebral artery ischemic stroke resulted in significant neurogenesis with progenitor cell migration into the dentate gyrus and subventricular areas. Poulsen, et.al. (2005) not only showed that IGF1 promoted neurogenesis, but antagonists of glutamate receptors did as well. Given these results and considered together with the previous discussion of arginine and nitric oxide, a post-stroke protocol that increases nitric oxide synthesis and blocks the NMDA receptor such as high dose arginine should be implemented together with human growth hormone and physical exercise. This is beginning to sound more and more like a prescription for a post-brain injury rehabilitation program. So why aren’t we doing this?

MINOCYCLINE
Now let’s add more data. Recently, a report aired on CNN and many other news programs that minocycline improved stroke outcomes. This was sparked by a study out of  Israel published in Neurology (Lampl, et.al. 2007) showing a reduction in neurologic deficits in humans following CVA injury as determined by the NIH Stroke Scale, the modified Rankin Scale and the Barthel Index when minocycline was administered within the early 6-24 hour cerebral infarct period. This was an open label, evaluator-blinded study of 152 patients. Additional data supporting this minocycline effect, which is characterized by a suppression of microglia activation and subsequent inflammatory reaction, comes from a study in the department of neurological surgery at University of California at San Francisco (Liu, et.al., 2007) that demonstrated that minocycline administered for 6 weeks post experimentally-induced middle cerebral artery ischemia in rats did not reduce infarct size, but did increase the number of newborn neurons. In addition, results showed improved motor coordination, decreased footfalls in the affected limb and decreased the preferential use of the unaffected limb during motor tasks, and increased learning and memory in spatial tasks,  In vitro studies (Chechniva, et.al. 2006: Ekdahl, et.al. 2003; Fan, et.al. 2005) have also demonstrated that inhibiting inflammatory activation of microglia and the upregulation of pro-inflammatory cytokines by indomethacin or minocycline resulted in a restoration of neurogenesis. The Fan, et.al. study went on to postulate that because minocycline inhibited the white matter damage by a lipopolysaccharide endotoxin by suppression of the microglial activation, it might be an effective therapeutic treatment for sepsis-induced neuron damage. Now we have another addition to our stroke protocol. Together with the neuroprotective effects and neurogenesis stimulus provided by growth hormone and IGF1 and arginine and exercise, minocycline decreases the post-stroke inflammation and thereby promotes neurogenesis, and in a well-controlled recent study reduces neurologic deficits following strokes in humans. So why aren’t we doing this?

 MEMANTINE or NAMENDA
But let’s look further. About 10 years ago I started using amantadine in my post-stroke patients. At that time, a structurally similar compound, memantine, was not approved by the FDA for use in the United States but had been used extensively in the rest of the world for neuroprotection for many years. In the past 3 years, however, memantine has been approved by the FDA for treatment of Alzheimer’s dementia. It doesn’t matter what the FDA approved it for. What matters is its mechanism of action. Much of this I have already discussed in the previous section on memantine, but allow me to add some salient points. Remember that its mechanism of action is to block the NMDA receptor thereby preventing the neurotoxic effects of aspartate and glutamate resulting in neuroprotection (Jain, 2000; Orgagozo, et.al. 2002; Mobius and Stoffler, 2002; Sang et.al., 2002; Szekely, et.al., 2002; Jones, et.al., 2001; Weber 2001; Suzuki, et.al 2001; Fisher, et.al., 2000; Parsons, et.al., 1999; Gorgulu, et.al. 2000). Remember that NMDA receptor antagonists also promote neurogenesis as discussed above. Therefore the post-stroke protocol must also include memantine. Why aren’t we doing this?

 PROZAC
Prozac or fluoxetine was discussed briefly in my abbreviated historical review of neurogenesis above. Let’s look more closely at the data. I referred to Dr. Duman’s study in Journal of Neuroscience (2000) that demonstrated stress induces hippocampal atrophy and neuronal loss and that antidepressant medications reverse it with increased cell proliferation and increased neuronal number. In a subsequent study (Malberg and Duman, 2003), they demonstrated that the decreased cell proliferation and neurogenesis with chronic stress was not the result of increased circulating corticosterone. However, it is important to make the following aside when discussing this study. While the neuronal loss was not the result of increased corticosterone levels, Huang and Herbert (2006) found that normal diurnal cortisol cycling was critical to the neurogenesis effect of fluoxetine and postulate that concurrent manipulation of this adrenal hormone with antidepressant medication might be additive to its neurogenic effect. A post stroke protocol must also evaluate the HPA axis as well. Why aren’t we doing this? 

MELATONIN
We have also discussed the neurotransmitter/hormone melatonin in previous sections and that it should be included in a post-stroke protocol. Let’s add more data.  Moriya, et.al. (2007) demonstrated that melatonin at pharmacologic doses exerts potent modulatory effect on the differentiation of neural stem cells during the proliferation phase and during the differentiation phase of the process, both increasing and decreasing neurogenesis respectively. Fernandez-Tresguerres Hernandez (2004) had presented data supporting these findings but added an additional interesting insight. There appears to be synergistic facilitation of neurogenesis by the combination of human growth hormone or IGF1 together with melatonin and estradiol. Of course, I had already included melatonin in the stroke protocol, but this finding that it may also promote differentiation of adult neural stem cells confirms melatonin to be an important addition. Why aren’t we doing this?

 THYROID
Thyroid hormone affects virtually every physiologic process in the body and it should not be at all surprising that it also plays a role in neurogenesis. Montero-Pedrazuela, et.al. (2006) have found that a short duration of hypothyroidism impairs cell proliferation in the dentate gyrus by as much as 30% as well as decreasing the number of new neuroblasts and immature neurons. The arborization of the dendrites in these few immature neurons was noted to be severely hypoplastic. These changes were reversed by treatment of chronic thyroid replacement. These results had also previously been demonstrated by Ambrogini, et.al. (2005) and Desouza, et.al. (2005) in adult rats and the latter study notes that the neural progenitor cells express thyroid receptor isoforms. These receptor isoforms were further characterized by Lemkine, et.al. (2005) as alpha TH receptors rather than beta isoforms. T3, not T4, was used in these studies as a ligand to characterize these receptors both in vitro and in vivo. These effects of thyroid hormone on neurogenesis are well established. In 1999, Gomes, et.al. found that T3 stimulates neurogenesis by two mechanisms. The first effect is by stimulating the formation of astrocytes and the second is by a direct effect on neural progenitors themselves. The astrocytes, these authors believe, secrete specific growth factors that in turn stimulate the neural progenitor cells to differentiate. This was observed in cerebellar granule cells which confirms a powerful primary and secondary influence of liothyronine ubiquitously in the central nervous system on adult neural stem cells.  Why aren’t we using this?

 SUMMARY
There appears to be a complex synergy among serotonin and other substances that have always been thought of as traditional neurotransmitters and pituitary and steroid and adrenal hormones in the promotion and regulation of neurogenesis in the adult central nervous system. McEwen (1996) states this well as follows: 

• “1. The hippocampus is an important brain structure for working and spatial memory in animals and humans, and it is also a vulnerable as well as plastic brain structure as far as sensitivity to epilepsy, ischemia, head trauma, stress, and aging. 
•  2. The hippocampus is also a target brain area for the actions of hormones of the steroid/thyroid hormone family, which traditionally have been thought to work by regulating gene expression. “Genomic” actions of steroid hormones involve intracellular receptors, whereas “nongenomic” effects of steroids involve putative cell surface receptors. Although this distinction is valid, it does not go far enough in addressing the variety of mechanisms that steroid hormones use to produce their effects on cells. This is because cell surface receptors may signal changes in gene expression, while genomic actions sometimes affect neuronal excitability, often doing so quite rapidly. 
•  3. Moreover, steroid hormones and neurotransmitters may operate together to produce effects, and sometimes these effects involve collaborations between groups of neurons. For example, a number of steroid actions in the hippocampus involve the coparticipation of excitatory amino acids. These interactions are evident for the regulation of synaptogenesis by estradiol in the CA1 pyramidal neurons of hippocampus and for the induction of dendritic atrophy of CA3 neurons by repeated stress as well as by glucocorticoid injections. In addition, neurogenesis in the adult and developing dentate gyrus is “contained” by adrenal steroids as well as by excitatory amino acids. In each of these three examples, NMDA receptors are involved. 
• 4. These results not only point to a high degree of interdependency between certain neurotransmitters and the actions of steroid hormones, but also emphasize the degree to which structural plasticity is an important aspect of steroid hormone action in the adult as well as developing nervous system.”

In the adult, supposedly fully developed brain, there exist neural stem cells which, when stimulated, are capable of differentiation into neurons that can be integrated into the functional neuronal network. The brain, like all other organs in the body, has now been shown to be able to heal itself after injury. The challenge to scientists and physicians is to identify and elucidate the chemicals and drugs and their interactions that will promote this healing process. 

As of this writing, the current management for a patient coming into the ER with a stroke is to get a CT scan of the brain, get an EKG, perform an entire cardiac workup to make sure the stroke wasn’t caused by a heart problem, tell the patient he had a stroke and send him to the rehabilitation unit where he will get physical, occupational and speech therapies. No thought is given to utilizing any of the medications discussed above, or to implementing any of the enormous amount of data published every day in the neuroscience and neurobiology journals. This is appalling to me as I consider that at stake is the paralysis and loss of speech of a human being which might be prevented. Why aren’t we doing this?  To continue with early 20^th century medical management of brain injury and stroke in the 21^st century is unconscionable.

Stay tuned, there’s more to come on my neurogenesis sermon.