The spinal cord can regenerate

Needle mice are able to repair damaged spinal cord

Ekaterina Gracheva, "Elements"

Fig. 1. As shown in the work under discussion, needle mice, in particular the Cairo mouse (Acomys cahirinus), are capable of regenerating damaged spinal cord. When it breaks in the area of the eighth thoracic vertebra (lesion site at T8), the extracellular matrix is remodeled (ECM remodelling), the number of keratansulfated proteoglycans (KSPG, keratansulfate proteoglycans) increases, and the level of the enzyme b-1,3-N-acetylglucosamintransferase 7, which controls the formation of these molecules, increases. Fibroblasts (fibrosis, fibrosis) and astrocytes (astroglyosis, astrogliosis) do not accumulate in the area of damage. House mice (Mus musculus) are not capable of regenerating such damage. Fibroblasts and astrocytes accumulate at the site of spinal cord injury. Fibroblasts (fibroblasts) are indicated in orange, astrocytes (astrocytes) — green. A drawing from the discussed article in the Developmental Cell.

Regeneration of organs and tissues is an asterisk task for adults of most mammalian species, including humans. Their organisms are able to heal a small wound on their own, but with age, even simple scratches leave scars. More serious and chronic damage leads to fibrosis — the proliferation of non-functional connective tissue. But some mammals have very extraordinary regenerative abilities. One example is the Cairo mice (Acomys cahirinus), related to needle mice. They are able not only to heal the skin, but also to completely restore damaged organs, including the spinal cord. A recent study has shown that this is probably due to the special organization of the extracellular matrix, which simultaneously prevents the formation of a fibrous scar and promotes the restoration of functional cells, in particular neurons.

Complete regeneration of organs and tissues is the ability of many animals (from sponges to amphibians), but most mammalian species are able to fully regenerate only individual tissues, such as the lining of the intestine and liver tissue. Even the skin with sufficiently large injuries (like a deep scratch several centimeters long) does not recover completely: a scar is formed, which is less elastic and sensitive than real skin. With more complex bodies, things are even sadder from the point of view of restoration: as a rule, the damaged tissue is replaced with a connective one, which cannot work as it is necessary for the normal functioning of the organ.

What happens when it gets damaged? Many cells of different types are activated in the damaged area. If, for example, you suddenly cut your finger, then platelets will not only prevent bleeding, but also activate cells that trigger and maintain inflammation. The same signals are sent to these cells by the damaged epithelium and nerve endings. Mast cells and neutrophils clean the wound from dead cells and bacteria trapped in it. Myofibroblasts (one of the connective tissue cells) begin to secrete the protein collagen, which allows the wound to close. Later, keratinocytes and epithelial cells are activated, covering the wound and forming a scar (for more information about this process, see in a review by C. D. Marshall et al., 2018. Cutaneous Scarring: Basic Science, Current Treatments, and Future Directions).

Ideally, myofibroblasts and immune cells should leave the site of damage after recovery, and the extracellular matrix (that is, molecules that support cells in the tissue) in the area of damage should gradually recover to normal. However, complete recovery in mammals is possible only if the fetus is damaged — in this case, no scars are formed. In adult mammals, except in exceptional cases, for example, skin restoration in wintering baribal bears (P. A. Iaizzo et al., 2012. Wound healing during hibernation by black bears (Ursus americanus) in the wild: elicitation of reduced scar formation), scars remain for a long time or even forever, especially after major damage. Scar tissue consists mainly of type I collagen (unlike type III collagen, which makes up the bulk of the extracellular matrix of the skin).

Scars are not only a change in the appearance of the skin or other organ, but also a violation of their function. The accumulation of collagen fibers cannot replace either muscle cells, changing the extensibility of organs such as the uterus or heart, or neurons of the spinal cord and brain, leading to a violation of cognitive, motor and other functions. If additional collagen—producing fibroblasts have not left the site of injury, the scar tissue can grow and turn into fibromas.

But there are mammals in which, even in adulthood, the damage is restored without the formation of scars. Needle mice (Acomys) are a genus of small rodents that live in the Middle East, South Asia and Africa. They got their name from the stiff hairs on the back, which resemble small needles, but do not really help in protecting against predators. In 2012, researchers from the USA and Kenya published a paper describing an interesting defense strategy against attack in needle mice (A. W. Seifert et al., 2012. Skin shedding and tissue regeneration in African spiny mice (Acomys)). It turned out that with rough treatment or attack, the skin of these animals easily tears and peels off, allowing the mouse to escape and leaving the enemy with nothing. However, even after serious wounds, the skin of needle mice is completely restored, including wool, nerve endings, skin glands and underlying muscle and adipose tissue (Fig. 2).

Fig. 2. Needle mice A. kempi (a) and A. percivali (b). Pay attention to the stiff, needle-like hairs on the animals' backs. c is an individual A. kempi after losing the skin on the back. d, e — skin restoration. After 3 days (d) the crusts on the wound are visible, after 30 days (e) the wounds are completely overgrown. f — new stiff hairs are visible on the damaged area of a needle mouse caught in the wild. The length of the scale segments is 1 cm . Figure from the article by A. W. Seifert et al., 2012. Skin shedding and tissue regeneration in African spiny mice (Acomys)

Skinks and geckos use a similar defense strategy, but they (unlike needle mice) the skin has a feature — the dermis is divided into two layers: a fragile outer layer and a more durable inner one. There is a layer of loose connective tissue between them. This is how pre-formed rupture sites are formed (A.M. Bauer et al., 1989. Mechanical Properties and Morphological Correlates of Fragile Skin in Gekkonid Lizards). Needle mice do not have such a feature. Their skin is similar in the composition of the extracellular matrix to the skin of ordinary mice, but less elastic, probably due to the fact that their glands and hair follicles are larger than those of mice. When the wound is overgrown, the scar tissue in needle mice consists mainly of collagen III, and not collagen I (as in other mammals), which may help the skin regenerate faster, and the scar tissue subsequently disappear.

Scientists comparing regeneration in needle-like and ordinary mice have shown that the inflammatory process is not so intense in needle-like mice. Proinflammatory factors and inflammation—related cells (neutrophils and some types of macrophages) are less pronounced in the wounds of needle mice, and factors associated with tissue repair are the opposite (J. Simkin et al., 2017. Macrophages are necessary for epimorphic regeneration in African spiny mice). Needle mice can also repair muscle tissue after injury, as well as exposure to myotoxins (M. Maden, J. O. Brant, 2018. Insights into the regeneration of skin from Acomys, the spiny mouse).

In recent years, scientists have found out that the internal organs of needle mice are also capable of regeneration. The last stage of many forms of kidney disease is tubulointerstitial fibrosis, which leads to complete organ failure and possible death of the patient. Healthy kidney parenchyma and renal tubules are replaced by connective tissue. But if we simulate such a condition on needle-like ones, then fibrous formations do not form in them, and damaged renal tubules retain their structure (D. M. Okamura et al., 2021. Spiny mice activate unique transcriptional programs after severe kidney injury regenerating organ function without fibrosis).

What about damage to the nervous system? After all, if needle mice are able to restore internal organs, then it can be assumed that they can also restore, for example, the spinal cord. In "ordinary" mammals, when the spinal cord is damaged, immune cells are activated that cause inflammation (including microglia). Astrocytes and spinal cord stromal cells secrete extracellular proteins (e.g. collagen IV) that form a fibrous scar (G. M. Cregg et al., 2014. Functional regeneration beyond the glial scar). Such a scar is a physical and chemical barrier to repair damaged axons.

In 2019, the first work devoted to the search for an answer to this question was published (K. A. Streeter et al., 2019. Molecular and histologic outcomes following spinal cord injury in spiny mice, Acomys cahirinus). Its authors damaged the spinal cord of needle and house mice by squeezing — such an operation simulates the most common spinal cord injury in humans. After a couple of days, the needle-like mice showed signs of restoring the function of the bladder: they could empty it on their own. House mice at the same time needed the help of vivarium technicians. Three days after the injury, house mice compared to needle mice had more active genes that are associated with wound formation (inflammation and tissue changes). In needle mice, genes whose products are involved in the restoration of nerve cells were activated (Fig. 3).

Fig. 3. Restoration of the spinal cord of brownies and needle mice after compression. House mice (a–c) have an accumulation of collagen IV (ColIV, red) in the area of damage, whereas needle mice (d–f) accumulate the main protein of myelin (MBP, myelin basic protein, green). a and d are longitudinal sections of the spinal cord; b, c, e, f are enlarged images of the injury site. The cores (DAPI) are colored blue. Figure from the article by K. A. Streeter et al., 2019. Molecular and histologic outcomes following spinal cord injury in spiny mice, Acomys cahirinus

Microscopic examination revealed that collagen IV, which forms most of the scar, does not accumulate at the site of injury in needle mice. But they have (but not house mice) myelin basic protein (MBP, myelin basic protein) is present there. This protein participates in axon regeneration (Z. Yan et al., 2021. Myelin basic protein enhances axonal regeneration from neural progenitor cells).

Research on regeneration in needle mice continues, and an article was published in the February issue of the journal Developmental Cell, which shows that needle mice are able to regenerate not just damaged, but severed spinal cord. After spinal cord severing surgery, the mice were monitored for 8 weeks. Their recovery was assessed on the Basso scale for mice (D. M. Basso et al., 2006. Basso Mouse Scale for Locomotion Detects Differences in Recovery after Spinal Cord Injury in Five Common Mouse Strains): 0 — ankle does not move, 9 — full mobility. In house mice, even after 8 weeks, the index did not reach 1. In needle mice, after two weeks, the index reached an average value of 2 (the ankle moved), and after 8 weeks the average index was 4 (mice could stand using their hind legs). 8 out of 14 experimental needle mice after 8 weeks could not only stand using all four paws, but also walk.

In addition, needle mice could control the work of the bladder after 3 weeks, whereas house mice had urinary incontinence and its retention in the bladder. Thus, needle mice are an almost unprecedented example of an adult mammal that can repair a damaged spinal cord.

Fig. 4. a — needle mouse (Acomys cahirinus). b — experiment plan: brownies and needle mice were prepared for the experiment (habituation) for 1 week, then the spinal cord was damaged (SCI, spinal cord injury). For 8 weeks after the operation, the animals were monitored, assessing their recovery on the Basso scale, as well as how the animals owned the bladder. After that, the animals were euthanized (euthanasia). c — motor activity of the house mouse and needle mouse 8 weeks after surgery. d — recovery score on the Basso scale (BMS score). Brownies (dark gray circles, Mus sham) and needle mice (dark blue circles, Acomys sham) who underwent a fictitious surgical intervention (the operation was performed, but the spinal cord was not cut) received maximum points on the scale. Light gray circles show values for mice after injury (Mus SCI), light blue squares - for needle mice (Acomys SCI). At week 8 of the experiment, parts of the needle mice were repeatedly severed the spinal cord (2x injury) to show that it is the restoration of the spinal cord that leads to the restoration of mobility. e is the percentage of animals that have regained control of the bladder (% of animals with blade control). f — urinary spots of brownie (Mus) and needle (Acomys) mice after spinal cord injury (SCI) or sham surgery (sham). Pay attention to the small spots and splashes of urine in house mice after spinal cord injury — they indicate that the injured mice do not control urination. Figure from the discussed article in Developmental Cell

What is the secret of needle mice? After 8 weeks, a large scar formed at the site of the injury in house mice, extending beyond the contour of the ordinary spinal cord. The needle mice had a much smaller scar. In the scar tissue of house mice, there were absolutely no signs of the growth of new axons (their axons were generally retracted back). In needle mice, on the contrary, regenerating axons stretched through the scar tissue.

There are ascending and descending nerve pathways in the spinal cord. The former carry information from organs and tissues, the latter deliver information to them. The researchers found that axons of both types of pathways were restored, and they formed synapses and conduction was observed in them.

Of course, there must be some molecular reasons for such an extraordinary regeneration. To identify them, the authors compared the genes that are expressed at the site of injury in brownie and needle mice. The biggest differences were in the signaling pathways associated with inflammation, as well as the activity of acetylglucosamintransferase enzymes. The fact that the activity of inflammatory processes decreases in needle mice has been shown by other authors. However, changing the work of acetylglucosamintransferases is a very interesting finding. Such enzymes affect the synthesis of the main glycosaminoglycans — polysaccharides, which, joining proteins, form proteoglycans, which make up most of the extracellular matrix. In particular, the level of enzymes that affect the formation of keratan sulfate and heparan sulfate proteoglycans changed in needle mice. The level of b-1,3-N-acetylglucosamintransferase 7, necessary for the synthesis of keratan sulfate proteoglycans, increased at the site of damage, while the level of N-deacetyl-N-sulfotransferases 3 and 4, modifying already formed heparan sulfate proteoglycans, on the contrary fell. Why do we need such changes?

B-1,3-N-acetylglucosamintransferase 7 synthesized at the site of injury creates an accumulation of ketanate sulfate proteoglycans on both sides of the site of injury. As expected, the level of heparasulfate proteoglycans, on the contrary, decreased. Such changes in the composition of the extracellular matrix could affect the restoration of axons. However, it is impossible to verify this directly. Therefore, the authors used an alternative methodology. They obtained Chinese hamster cells of the CHO line, which produced b-1,3-N-acetylglucosamintransferase of 7 needle mice, and then grew neurons of the ganglia of the posterior roots and the cerebral cortex of mice on these cells. In both types of neurons, compared with the control, the processes (dendrites and axons) grew more efficiently on such modified cells (Fig. 5).

Fig. 5. The extracellular matrix affects the repair of neurons. a — longitudinal section of spinal cord injury in house mice (Mus) and needle mice (Acomys) 8 weeks after injury. The tissue is stained with antibodies to keratan sulfated proteoglycans (KSPG). White arrows indicate the deposition of these molecules in the area of damage. To detect the presence of keratan-sulfated proteoglycans, tissue samples were also incubated with keratanase, which destroys these molecules. The intensity of the KSPG signal decreased. b is the corresponding numerical data. c is a longitudinal section of spinal cord injury in house mice (Mus) and needle mice (Acomys) 8 weeks after the injury. The tissue is stained with antibodies to heparan sulfated proteoglycans (HSPG). To detect the presence of heparan sulfated proteoglycans, the samples were also incubated with heparinase III (Heparinase), which destroys these molecules. The intensity of the HSPG signal decreased. d is the corresponding numerical data. e — scheme of an experiment on the effect of b-1,3-N-acetylglucosamintransferase 7 on the growth of neuronal processes. The green color indicates the cells of the CHO line expressing this enzyme (Transfected CHO cells); the black color indicates the neurons of the ganglia of the posterior roots and the cerebral cortex of mice (Mus DRG or cortical neurons). On the first day of the experiment, CHO cells were planted, transfection (introduction of DNA encoding the gene b-1,3-N-acetylglucosamintransferase 7 (B3gnt7-GFP) or green fluorescent protein (GFP)) was carried out on the third day. On the sixth day, neurons were planted on these cells, and on the seventh day, the experiment was stopped and the length of their processes was measured. f is a typical type of neurons on CHO cells expressing B3gnt7-GFP or GFP. The yellow color indicates β-tubulin III, a marker of growing nerve processes. g, h, i — graphs of the total length of the processes of neurons (total neurite length), the length of axons (axon length) and the number of branches (n of braches) to the distance to the cell body (distance from soma); lengths were measured in microns. Figure from the discussed article in Developmental Cell

Thus, the probable secret of regeneration of needle mice is the structure of the extracellular matrix and its response to damage. Of course, this is not the only factor — the role of inflammation management is difficult to underestimate. However, the extracellular matrix remains a poorly understood element of this process. His device, of course, organizes the process, provides the correct microenvironment for incoming cells, promotes the exchange of chemicals between them. But these relationships are so complex that scientists will have to deal with them for a long time. Interestingly, the results of this study contradict previous publications, which claimed that keratan sulfate proteoglycans reduce the intensity of axon regeneration (S. Imatama et al., 2011. Keratan Sulfate Restricts Neural Plasticity after Spinal Cord Injury).

It is also interesting why and how exactly needle mice, and not brownies or, say, their close relatives gerbils, acquired such amazing regeneration abilities. Perhaps someday we will get an answer to it.

A source: Nogueira-Rodrigues et al., Rewired glycosylation activity promotes scarless regeneration and functional recovery in spiny mice after complete spinal cord transection // Developmental Cell. 2022.

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