Scientists at UC Irvine’s Reeve-Irvine Research Center have discovered that injections of salmon fibrin injections into the injured spinal cord plus injections of a gene inhibitor into the brain restored voluntary motor function impaired by spinal cord injury.
Gail Lewandowski and Oswald Steward, director of the Reeve-Irvine Research Center at UCI, examined rodents that had received spinal cord injuries. They were able to heal the damage by developmentally turning back the clock in a molecular pathway that is critical to the formation of the corticospinal nerve tract, and by providing a scaffold for the growing neurons so that the axons of these growing neurons could grow and make the necessary connections with other cells. Their research was published in the July 23 issue of The Journal of Neuroscience.
The work of Steward and Lewandowski is an extension of previous research at UC Irvine from 2010. Steward and his colleagues discovered that the axon of neurons grow quite well once an enzyme called PTEN is removed from the cells. PTEN is short for “phosphatase and tensin homolog,” and it removes phosphate groups from specific proteins and lipids. In doing so, PTEN signals to cells to stop dividing and it can also direct cells to undergo programmed cell death (a kind of self-destruct program). PTEN also prevents damaged tissues from regenerating sometimes, because it is a protein that puts the brakes of cell division. Mutations in PTEN are common in certain cancers, but the down-regulation of PTEN is required for severed axons to re-form, extend, migrate to their original site, and form new connections with their target cells.
After two years, team from U.C. Irvine discovered that injections of salmon fibrin into the damaged spinal cord or rats filled cavities at the injury site and provided the axons with a scaffolding upon which they could grow, reconnect and facilitate recovery. Fibrin produced by the blood system when the blood vessels are breached and it is a fibrous, insoluble protein produced by the blood clotting process. Surgeons even use it as a kind of surgical glue.
“This is a major next step in our effort to identify treatments that restore functional losses suffered by those with spinal cord injury,” said Steward, professor of anatomy & neurobiology and director of the Reeve-Irvine Research Center. “Paralysis and loss of function from spinal cord injury has been considered irreversible, but our discovery points the way toward a potential therapy to induce regeneration of nerve connections.”
In their study, Steward and Lewandowski subjected rats to spinal cord injuries, and then assessed their defects. Because these were upper back injuries, the rats all showed impaired forelimb (hand) movement. Steward and Lewandowski then treated these animals with a combination of salmon fibrin at the site of injury and a modified virus that made a molecule that inhibited PTEN. These viruses were genetically engineered adenovirus-associated viruses encoded a small RNA that inhibited translation of the PTEN gene (AAVshPTEN). This greatly decreased the levels of PTEN protein in the neurons. Other rodents received control treatments of only AAVshPTEN and no salmon fibrin.
The results were remarkable. Those rats that received the PTEN inhibitor alone showed no improvement in their forelimb function, but those animals who were given AAVshPTEN plus the salmon fibrin recovered forelimb use (at least reaching and grasping).
“The data suggest that the combination of PTEN deletion and salmon fibrin injection into the lesion can significantly enhance motor skills by enabling regenerative growth of corticospinal tract axons,” Steward said.
Statistics compiled by the Christopher & Dana Reeve Foundation suggests that approximately 2 percent of Americans have some form of paralysis that is the result of a spinal cord injury. Spinal cord injuries break connections between nerves and muscles or nerves and other nerves. Even injuries the size of a grape can cause complete loss of function below the level of the injury. Injuries to the neck can cause paralysis of the arms and legs, an absence of sensation below the shoulders, bladder and bowel incontinence, sexual dysfunction, and secondary health risks such as susceptibility to urinary tract infections, pressure sores and blood clots due to an inability to move one’s legs.
Steward said the next objective is to learn how long after injury this combination treatment can be effectively administered. “It would be a huge step if it could be delivered in the chronic period weeks and months after an injury, but we need to determine this before we can engage in clinical trials,” he said.
A host of preclinical studies have examined the ability of stem cells to improve the condition of laboratory animals that have suffered a spinal cord injury. While these studies vary in their size, design, and quality, there has been little cumulative analysis of the data generated by these studies.
Fortunately, there is a powerful analytical tool that can examine data from many studies and this type of analysis is called a “meta-analysis.” Meta-analyses use sophisticated statistical packages to systematically reassess a compilation of the data contained within these papers. Meta-analyses are exhausting, but potentially very useful. Such a meta-analysis is also very important because it provides researchers with an indication of what problems must be worked out before these treatments advance to human clinical trials and what aspects of the treatment work better than others.
A recent meta-analysis of stem cell therapy on animal models of spinal cord injury has been published by Ana Antonic, MSc, David Howells, Ph.D., and colleagues from the Florey Institute and the University of Melbourne, Australia, along with Malcolm MacLeod and colleagues from the University of Edinburgh, UK in the open access journal PLOS Biology.
The goal of regenerative spinal cord treatments is to use stem cells to replace dead cells within damaged areas of the spinal cord. Such treatments would be given to spinal cord injury patients in the hope of improving the ability to move and to feel below the site of the injury. Many experiments that utilize animal models of spinal cord injury have used stem cells to treat laboratory animals that have suffered spinal cord injury, but, unfortunately, these studies are limited in scale by size (as a result of financial considerations), practical and ethical considerations. Such limitations hamper each individual study’s statistical power to detect the true effects of the stem cell implantation. Also, these studies use different types of stem cells in their treatment scenarios, inject those cells differently induce spinal cord injuries differently, and test their animals for functional recovery differently.
To assess these studies, this new paper examined 156 published studies, all of which tested the effects of stem cell treatments on about 6,000 spinal cord-injured animals.
Overall, they found that stem cell treatment results in an average improvement of about 25 percent over the post-injury performance in both sensory (ability to feel) and motor (ability to move) outcomes. Unfortunately, the variation from one animal to another varied widely.
For sensory outcomes the degree of improvement tended to increase with the number of cells implanted. Such dose-responsive results tend to indicate that the improvements are actually due to the stem cells, and therefore, this stem cell-mediated effect represents a genuine biological effect.
The authors also measured the effects of bias. Simply put, if the experimenters knew which animals were treated and which were untreated, then they might be more likely to report improvements in the stem cell-treated animals. They also examined the way that the stem cells were cultured, the way that the spinal injury was generated and the way that outcomes were measured. In each case, important lessons were learned that should help inform and refine the design of future animal studies.
The meta-analysis also revealed some surprises that should provoke further investigations. For example, there was little evidence that female animals showed any beneficial sensory effects as a result of stem cell treatments. Also, the efficacy of the stem cell treatment seemed to not depend on whether immunosuppressive drugs were administered or not.
The authors conclude, “Extensive recent preclinical literature suggests that stem cell-based therapies may offer promise; however the impact of compromised internal validity and publication bias means that efficacy is likely to be somewhat lower than reported here.”
Even though human clinical trials are in the works, such trials will continue to be informed by preclinical studies on laboratory animals.
After injury to the spinal cord, glial cells and neural stem cells in the spinal cord contribute to the formation of the “glial scar.” This glial scar is rich in molecules known as chondroitin sulfate proteoglycans (CSPGs) that are known to repel growing axons. Therefore, the glial scar is viewed as a major impediment to spinal cord regeneration.
However, new work from the Karolinska Institutet in Solna, Sweden has confirmed that the glial scar actually works to contain the damage within the spinal cord. Far from impairing spinal cord recovery, the stem cell-mediated formation of the glial scar confines the damage to a discrete portion of the spinal cord and prevents it from spreading.
Trauma to the spinal cord can sever those nerve fibers that conduct nerve impulses to from the brain to skeletal muscles below the level of spinal cord injury. Depending on where the spinal cord is injured and the severity of the injury, spinal cord injuries can lead to a various degrees of paralysis. Such paralysis is often permanent, since the severed nerves do not grow back.
The absence of neural regeneration required an explanation, since cultured neurons whose axons are severed can regenerate both in culture and in a living creatures (for an excellent review, see Nishio T. Axonal regeneration and neural network reconstruction in mammalian CNS. J Neurol. 2009 Aug;256 Suppl 3:306-9). Thus, neuroscientists have concluded that the injured spinal contains a variety of molecules that inhibit axonal outgrowth and regeneration.
This hypothesis has been demonstrated since many axon growth inhibitors have been isolated from the injured spinal cord (see Schwab ME (2002) Repairing the injured spinal cord. Science 295:1029–1031). Such molecules include proteins like Nogo, Myelin-Associated Glycoprotein (MAG), and Oligodendrocyte-Myelin Glycoprotein (OMgp). However, as the Nishio review points out, axons from severed nerved have been seen growing throughout the central nervous system. Therefore, most of the blame for a lack of regrowth has been pinned on the glial scar.
A new study by Jonas Frisén of the Department of Cell and Molecular Biology and his colleagues has shown that the neural stem cell population in the spinal cord are the main contributors to the glial scar. However, when glial scar formation was prevented after spinal cord injury, the injured area in the spinal cord expanded and more nerve fibers were severed. Furthermore, in their mouse model, a great number of nerve cells died in those mice that did not make glial scars when compared to those mice that were able to produce a normal glial scar.
“It turned out that scarring from stem cells was necessary for stabilizing the injury and preventing it from spreading,” said Frisén. “Scar tissue also facilitated the survival of damaged nerve cells. Our results suggest that more rather than less stem cell scarring could limit the consequences of a spinal cord injury.”
According to earlier animal studies, recovery can be improved by transplanting stem cells to the injured spinal cord. These new findings suggest that stimulating the spinal cord’s own stem cells could offer an alternative to cell transplantation therapies.
This paper appeared in the journal Science, 1 November 2013: 637-640, and the first author was Hanna Sabelström. This interesting paper might be leaving one thing out when it comes to spinal cord regeneration. Once the acute phase of spinal cord injury is completed and the chronic phase begins, the glial scar does in fact prevent spinal cord regeneration. This is the main reason Chinese researchers have used chondroitinase enzymes to digest the scar in combination with transplantations on stem cells. By weakening the repulsive effects of the glial scar, these stem cells can form axons that grow through the scar. Also, olfactory ensheathing cells or OECs seem to be able to shepherd axons through the scar, although the degree of regeneration with these cells has been modest, but definitely real. Therefore, negotiating axonal regeneration through the glial scar remains a major challenge of spinal cord injury. Thus, while the glial scar definitely has short-term benefits, for the purposes or long-term regeneration, it is a barrier all the same.
A gaggle of laboratory animal experiments and clinical studies in human patients have established that stem cell injections into the spinal cord after spinal cord injury promote functional recovery (see Beattie, M. S., et al., Exp. Neurol. 148(2):453‐463; 1997; Bennett, D. L., et al., J. Neurosci. 20(1):427‐437; 2000; Kim HK, et al., PLos One 4(3): e4987 2009; Lu, P.; Tuszynski, M. H. Exp. Neurol. 209(2):313‐320; 2008; McTigue, D. M., et al., J. Neurosci. 18(14):5354-5365; 1998; Widenfalk, J.; Lundströmer, K. J. Neurosci. 21(10):3457‐3475; 2001; also see Salazar DL, et al., PLoS ONE, August 2010; Hooshmand M, et al., PLoS ONE, June 2009; Cummings BJ, et al., Neurological Research, July 2006; and Cummings BJ, et al., PNAS, September 19, 2005). Stem Cell, Inc., for example, has conducted several tests with human patients using their HuCNS-SC human neural stem cell line, and transplantation of these stem cells promotes functional recovery in human patients who have suffered spinal cord injury.
However, one factor that has yet to be properly determined is the best site for stem cell injection. Previous work by scientists at the Keio University School of Medicine in Japan has shown that injection of neural stem cells and neural progenitor cells (NS/PCs) into non-injured sites by either intravenous or intrathecal (introduced directly into the space under the arachnoid membrane of the brain or spinal cord) administration failed to produce sufficient engraftment of stem cells at the site of injury.
Instead cells were trapped in the lungs and kidneys, and many mice even developed fatal lung conditions as a result of intravenous administration (see Takahashi Y., et al., Cell Transplant. 2011;20(5):727-39). These data convinced them that intralesional application of the stem cells (injections directly into the damaged site of the spinal cord) might be the most effective and reliable method for NS/PC tranplantations.
A new study by the Keio group has attempted to ascertain the efficacy of the intralesional injections. Mice with spinal cord injuries were injected with NS/PCs that had been derived from mice that expression glowing proteins. This allowed the injected cells to be tracked with bio-luminescence imaging (BLI).
The principal investigator of this research is Masaya Nakamura from the Department of Orthopedic Surgery at the Keio University School of Medicine. Dr. Nakamura and his team gave mice spinal contusions at the level of the tenth thoracic vertebra. Then some mice were given low doses and others high doses of NS/PCs that were derived from fetal mice (for those who are interested, low dose – 250,000 cells per mouse; high dose – 1 million cells per mouse) nine days after spinal cord injury. These mice were further divided into two groups: those injected at the lesion epicenter (E), those injected at sites at the front and back of the lesion (RC for “rostral/caudal”). Thus there were four groups total: High dose E, High dose RC, Low dose E, and Low dose RC.
All four groups showed better functional recovery than the control group, which was injected with phosphate buffered saline. BLI showed that the number of cells that survived in each of the four cell-transplanted groups was about the same across these groups. Thus injecting more cells does not lead to greater numbers of surviving neural stem cells. This makes sense, since the damaged spinal cord in very inhospitable place for transplanted cells.
However, when the mice were examined for the expression of particular brain-derived neurotropic factors, the expression of such genes was higher in the RC-injected mice than in the E-injected mice. These results seems to explain why the transplanted NS/PCs differentiated more readily into neurons in the RC-injected mice rather than a type of glial cell known as an astrocyte, as was the case in the E-injected mice.
Nakamura and his team interpreted these results to mean that the environments of the E and RC sites can both support the survival of transplanted NS/PCs during the sub-acute phase of spinal cord injury. The authors conclude with a practical note: “Therefore, we conclude that it is optimal to graft a certain threshold number of NS/PCs into the epicenter lesion during the sub-acute phase of SCI, and thereby avoid causing further iatrogenic injury to the intact RC regions of the spinal cord.”
Hopefully Nakamura’s work will be translated into further human clinical trials. One feature of this study is that a particular threshold of stem cells survive when injected into the spinal cord and injecting larger numbers of cells does not increase the number of surviving cells. Injecting more cells might only contribute to the cell debris in the spinal cord. This is certainly a good thing to know when conducting clinical trials with neural stem cells in spinal cord-injured patients.
One of the challenges of stem cell-based therapies is cell survival. Once stem cells are implanted into a foreign site, many of them tend to pack up and die before they can do any good. For this reason, many scientists have examined strategies to improve stem cell survival.
A new technique that improves stem cells survival have been discovered by Yubo Fan and his colleagues at Beihang University School of Biological Science and Medical Engineering. This non-chemical technique, biphasic electrical stimulation (BES) might become important for spinal cord injury patients in the near future.
Spinal cord injury affects approximately 250,000 Americans, with 52% being paraplegic and 47% quadriplegic. There are 11,000 new spinal cord injuries each year and 82% are male.
Stem cell transplantions into the spinal cord to regenerate severed neurons and associated cells provides a potentially powerful treatment. However, once stem cells are implanted into the injured spinal cord, many of them die. Cell death is probably a consequence of several factors such as a local immune response, hypoxia (lack of oxygen), and probably most importantly, limited quantities of growth factors.
Fan said of his work, “We’ve shown for the very first time that BES may provide insight into preventing growth factor deprivation-triggered apoptosis in olfactory bulb precursor cells. These findings suggest that BES may thus be used as a strategy to improve cell survival and prevent cell apoptosis (programmed cell death) in stem cell-based transplantation therapies.”
Since electrical stimulation dramatically accelerates the speed of axonal regeneration and target innervation and positively modulates the functional recovery of injured nerves, Fan decided to test BES. His results showed that BES upregulated all the sorts of responses in stem cells that you would normally see with growth factors. Thus BES can increase stem cell survival without exogenous chemicals or genetic engineering.
Fan and his team examined the effects of BES on olfactory bulb neural precursor cells and they found that 12 hours of BES exposure protected cells from dying after growth factor deprivation. How did BES do this? Fan and other showed that BES stimulated a growth factor pathway called the PI3K/Akt signaling cascade. BES also increase the output of brain-derived neurotrophic factor.
“What was especially surprising and exciting,” said Fan, “was that a non-chemical procedure can prevent apoptosis in stem cell therapy for spinal cord patients.” Fan continued: “How BES precisely regulates the survival of exogenous stem cells is still unknown but will be an extremely novel area of research on spinal cord injury in the future.”
BES can improve the survival of neural precursor cells and will provide the survival of neural precursor cells and will provide the basis or future studies that could lead to novel therapies for patients with spinal cord injury.
Disclaimer: I am reporting on this experiment because of its significance for people with spinal cord-injuries even though I remain appalled at the manner in which the stem cells were acquired.
An international research team has reported that a single set of injections of human neural stem cells had provided significant neuronal regeneration and improvement of function in rats impaired by acute spinal cord injury.
Dr. Martin Marsala, who is professor of anesthesiology at the University of California, San Diego, with colleagues from academic institutions in Slovakia, the Czech Republic, and the Netherlands, used neural stem cells derived from an aborted human fetus to treat spinal cord-injured rats.
Sprague-Dawley rats received spinal cord injuries at the level of the third lumbar vertebra by means of compression. Such injuries render the rats incapable of using their hind legs. They cannot climb a ladder, walk a catwalk or perform other tasks that require the effective use of their hind legs.
The stem cells that were transplanted into the spinal cords of these rats were NSI-566RSC cells, which were provided by the biotechnology company Neuralstem. These cells were initially isolated from the spinal cord of an eight-week old human fetus whose life was terminated through elective abortion. These cells have been grown in culture and split many times. They are a neural stem cell culture that has the capacity to form neurons and glia.
The rats were broken into six groups, and four of these groups received spinal cord injuries. One of these spinal cord-injured groups received injections of were injured NSI-566RSC cells (12 injections total, about 20,000 cells per microliter of fluid injected), another received injections of only fluid, and the third group received no injections. The final spinal cord-injured group of rats received injections of NSI-566RSC cells that had been genetically engineered to express a green glowing protein. Another group of rats were operated on, but no spinal cord injury was given to these animals, and the final group of rats were never operated on.
All rats that received injections of cells were administered powerful drugs to prevent their immune systems from rejecting the administered human cells before the injections (methylprednisolone acetate for those who are interested at 10 mg / kg), and after the stem cell injections (tacrolimus at 1.5 mg / kg).
The results were significant and exciting. In the words of Marsala, “The primary benefits were improvement in the positioning and control of paws during walking tests and suppression of muscle spasticity.” Spasticity refers to an exaggerated muscle tone or uncontrolled spasms of muscles. Spasticity is a serious and common complication of traumatic injury. It can cause severe cramping and uncontrolled contractions of muscles, which increases the patient’s pain and decreases their control.
First, it is clear from several control experiments that the injection procedure did not affect the spinal cord function of these animals, since the sham injected rats had perfectly normal use of their hind limbs and normal sensory function of their limbs. Thus the injection procedure is innocuous. Also, the use of the drugs to suppress the immune response were also equally unimportant when it came to the spinal cord health of the rats.
Two months after the stem cell injections, the rats were subjected to the “catwalk test,” in which the animals walked a narrow path and their paw position was assessed. As you can see in the figure below, the stem cell-injected rats have a paw position that is far more similar to the normal rats than to the spinal cord injured rats.
Secondly, when muscle spasticity was measured, the stem cell-injected rats showed definite decreases in muscle spasticity. The spinal cord-injured rats that received no stem cell injections showed no such changes.
Sensory assessments also showed improvements in the stem cell-treated rats, but the improvements were not stellar. Nevertheless, the stem cell-treated rats progressively improved in their sensory sensitivity whereas the non-treated spinal cord-injured rats consistently showed no such improvement.
What were the implanted cells doing? To answer this question, Marsala and his co-workers examined tissue sections of spinal cords from the rats implanted with the glowing green stem cells. According to Marsala, the implanted neural stem cells are stimulating host neuron regeneration and partially replacing the function of lost neurons.
Marsala explained: “Grafted spinal stem cells are a rich source of different growth factors which can have a neuroprotective effect and can promote sprouting of nerve fibers of host neurons. We have demonstrated that grafted neurons can develop contacts with the host neurons and, to some extent, restore the connectivity between centers, above and below the injury, which are involved in motor and sensory processing.”
The implanted neural stem cells definitely showed extensive integration with the spinal nerves of the host rats. Again Marsala, “In all cell-grafted animals, there was a robust engraftment and neuronal maturation of grafted human neurons was noted.” Marsala continued: “Importantly cysts or cavities were not present in any cell-treated animal. The injury-caused cavity was completely filled by grafted cells.”
Marsala’s goal is to used a neuronal stem cell line derived from a patient-specific induced pluripotent stem cell line in a clinical trial. For now, the UC San Diego Institutional Review Board or IRB is reviewing a small phase 1 clinical trial to test the safety and efficacy of this neural stem cell line in patients with spinal cord injuries who have no feeling or motor function below the level of the spinal cord injury.