Keith K. Fenrich

Canadian Association of Neuroscience Review: Axonal Regeneration in the Peripheral and Central Nervous Systems – Current Issues and Advances

Injured nerves regenerate their axons in the peripheral (PNS) but not the central nervous system (CNS). The contrasting capacities have been attributed to the growth permissive Schwann cells in the PNS and the growth inhibitory environment of the oligodendrocytes in the CNS. In the current review, we first contrast the robust regenerative response of injured PNS neurons with the weak response of the CNS neurons, and the capacity of Schwann cells and not the oligodendrocytes to support axonal regeneration. We then consider the factors that limit axonal regeneration in both the PNS and CNS. Limiting factors in the PNS include slow regeneration of axons across the injury site, progressive decline in the regenerative capacity of axotomized neurons (chronic axotomy) and progressive failure of denervated Schwann cells to support axonal regeneration (chronic denervation). In the CNS on the other hand, it is the poor regenerative response of neurons, the inhibitory proteins that are expressed by oligodendrocytes and act via a common receptor on CNS neurons, and the formation of the glial scar that prevent axonal regeneration in the CNS. Strategies to overcome these limitations in the PNS are considered in detail and contrasted with strategies in the CNS.

Long‐term in vivo imaging of normal and pathological mouse spinal cord with subcellular resolution using implanted glass windows

•  Chronic in vivo imaging of cellular interactions within the adult spinal cord with subcellular resolution is important for understanding cellular physiology and disease progression. •  Previous approaches for chronic in vivo spinal cord microscopy have required surgery for each imaging session. •  Here we describe a novel method for implanting glass windows over the exposed spinal cords of adult mice for repeated in vivo microscopy. •  We show that the windows remain clear for many months after implantation, do not damage axons or blood vessels, and are useful for studying cellular dynamics after spinal cord injury. •  Our method represents an original technical breakthrough for scientists involved in spinal cord research and in vivo imaging, and is a useful tool for studying cellular physiology and disease progression.

Pericytes impair capillary blood flow and motor function after chronic spinal cord injury

Blood vessels in the central nervous system (CNS) are controlled by neuronal activity. For example, widespread vessel constriction (vessel tone) is induced by brainstem neurons that release the monoamines serotonin and noradrenaline, and local vessel dilation is induced by glutamatergic neuron activity. Here we examined how vessel tone adapts to the loss of neuron-derived monoamines after spinal cord injury (SCI) in rats. We find that, months after the imposition of SCI, the spinal cord below the site of injury is in a chronic state of hypoxia owing to paradoxical excess activity of monoamine receptors (5-HT1) on pericytes, despite the absence of monoamines. This monoamine-receptor activity causes pericytes to locally constrict capillaries, which reduces blood flow to ischemic levels. Receptor activation in the absence of monoamines results from the production of trace amines (such as tryptamine) by pericytes that ectopically express the enzyme aromatic L-amino acid decarboxylase (AADC), which synthesizes trace amines directly from dietary amino acids (such as tryptophan). Inhibition of monoamine receptors or of AADC, or even an increase in inhaled oxygen, produces substantial relief from hypoxia and improves motoneuron and locomotor function after SCI.

Long- and short-term intravital imaging reveals differential spatiotemporal recruitment and function of myelomonocytic cells after spinal cord injury.

•  Inflammatory cells such as myelomonocytic cells are key players in the progression and recovery from spinal cord injury (SCI). •  However, the precise spatiotemporal distributions and roles of different subpopulations of myelomonocytic cells remain unclear in part because their dynamics have not been examined in vivo. •  Using chronic in vivo two‐photon microscopy techniques in adult transgenic mice with SCI, we show that infiltrating and resident myelomonocytic cells have differential spatiotemporal distribution patterns to injury sites. •  We also show that infiltrating myelomonocytic cells are associated with the collapse of certain cut axon terminals thus potentially impeding recovery, whereas resident myelomonocytic cells clear axon debris, which may be important for axon regrowth and recovery. •  These results set a framework to understand the roles of different subpopulations of myelomonocytic cells in SCI, and may be important for the development of therapies that target specific immune cell populations at precise times post‐injury.

Implanting Glass Spinal Cord Windows in Adult Mice with Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) in adult rodents is the standard experimental model for studying autonomic demyelinating diseases such as multiple sclerosis. Here we present a low-cost and reproducible glass window implantation protocol that is suitable for intravital microscopy and studying the dynamics of spinal cord cytoarchitecture with subcellular resolution in live adult mice with EAE. Briefly, we surgically expose the vertebrae T12-L2 and construct a chamber around the exposed vertebrae using a combination of cyanoacrylate and dental cement. A laminectomy is performed from T13 to L1, and a thin layer of transparent silicone elastomer is applied to the dorsal surface of the exposed spinal cord. A modified glass cover slip is implanted over the exposed spinal cord taking care that the glass does not directly contact the spinal cord. To reduce the infiltration of inflammatory cells between the window and spinal cord, anti-inflammatory treatment is administered every 2 days (as recommended by ethics committee) for the first 10 days after implantation. EAE is induced only 2-3 weeks after the cessation of anti-inflammatory treatment. Using this approach we successfully induced EAE in 87% of animals with implanted windows and, using Thy1-CFP-23 mice (blue axons in dorsal spinal cord), quantified axonal loss throughout EAE progression. Taken together, this protocol may be useful for studying the recruitment of various cell populations as well as their interaction dynamics, with subcellular resolution and for extended periods of time. This intravital imaging modality represents a valuable tool for developing therapeutic strategies to treat autoimmune demyelinating diseases such as EAE.

The role of cAMP and its downstream targets in neurite growth in the adult nervous system

Injured neurons in the adult mammalian central nervous system (CNS) have a very limited capacity for axonal regeneration and neurite outgrowth. This inability to grow new axons or to regrow injured axons is due to the presence of molecules that inhibit axonal growth, and age related changes in the neurons innate growth capabilities. Available levels of cAMP are thought to have an important role in linking both of these factors. Elevated levels of cAMP in the developing nervous system are important for the guidance and stability of growth cones. As the nervous system matures, cAMP levels decline and the growth promoting effects of cAMP diminish. It has frequently been demonstrated that increasing neuronal cAMP can enhance neurite growth and regeneration. Some methods used to increase cAMP include administration of cAMP agonists, conditioning lesions, or electrical stimulation. Furthermore, it has been proposed that multiple stages of cAMP induced growth exist, one directly caused by its downstream effector Protein Kinase A (PKA) and one caused by the eventual upregulation of gene transcription. Although the role cAMP in promoting axon growth is well accepted, the downstream pathways that mediate cAMP-mediated axonal growth are less clear. This is partly because various key studies that explored the link between PKA and axonal outgrowth relied on the PKA inhibitors KT5720 and H89. More recent studies have shown that both of these drugs are less specific than initially thought and can inhibit a number of other signalling molecules including the Exchange Protein Activated by cAMP (EPAC). Consequently, it has recently been shown that a number of intracellular signalling pathways previously attributed to PKA can now be attributed solely to activation of EPAC specific pathways, or the simultaneous co-activation of PKA and EPAC specific pathways. These new studies open the door to new potential treatments for repairing the injured spinal cord.

HB-GAM (pleiotrophin) reverses inhibition of neural regeneration by the CNS extracellular matrix

Chondroitin sulfate (CS) glycosaminoglycans inhibit regeneration in the adult central nervous system (CNS). We report here that HB-GAM (heparin-binding growth-associated molecule; also known as pleiotrophin), a CS-binding protein expressed at high levels in the developing CNS, reverses the role of the CS chains in neurite growth of CNS neurons in vitro from inhibition to activation. The CS-bound HB-GAM promotes neurite growth through binding to the cell surface proteoglycan glypican-2; furthermore, HB-GAM abrogates the CS ligand binding to the inhibitory receptor PTPσ (protein tyrosine phosphatase sigma). Our in vivo studies using two-photon imaging of CNS injuries support the in vitro studies and show that HB-GAM increases dendrite regeneration in the adult cerebral cortex and axonal regeneration in the adult spinal cord. Our findings may enable the development of novel therapies for CNS injuries.

Single pellet grasping following cervical spinal cord injury in adult rat using an automated full-time training robot

Task specific motor training is a common form of rehabilitation therapy in individuals with spinal cord injury (SCI). The single pellet grasping (SPG) task is a skilled forelimb motor task used to evaluate recovery of forelimb function in rodent models of SCI. The task requires animals to obtain food pellets located on a shelf beyond a slit at the front of an enclosure. Manually training and testing rats in the SPG task requires extensive time and often yields results with high outcome variability and small therapeutic windows (i.e., the difference between pre- and post-SCI success rates). Recent advances in automated SPG training using automated pellet presentation (APP) systems allow rats to train ad libitum 24h a day, 7 days a week. APP trained rats have improved success rates, require less researcher time, and have lower outcome variability compared to manually trained rats. However, it is unclear whether APP trained rats can perform the SPG task using the APP system after SCI. Here we show that rats with cervical SCI can successfully perform the SPG task using the APP system. We found that SCI rats with APP training performed significantly more attempts, had slightly lower and less variable final score success rates, and larger therapeutic windows than SCI rats with manual training. These results demonstrate that APP training has clear advantages over manual training for evaluating reaching performance of SCI rats and represents a new tool for investigating rehabilitative motor training following CNS injury.

Inhibiting cortical protein kinase A in spinal cord injured rats enhances efficacy of rehabilitative training.

Elevated levels of the second messenger molecule cyclic adenosine monophosphate (cAMP) are often associated with neuron sprouting and neurite extension (i.e., neuroplasticity). Phosphokinase A (PKA) is a prominent downstream target of cAMP that has been associated with neurite outgrowth. We hypothesized that rehabilitative motor training following spinal cord injuries promotes neuroplasticity via PKA activation. However, in two independent experiments, inhibition of cortical PKA using Rp-cAMPS throughout rehabilitative training robustly increased functional recovery and collateral sprouting of injured corticospinal tract axons, an indicator of neuroplasticity. Consistent with these in vivo findings, using cultured STHdh neurons, we found that Rp-cAMPS had no effect on the phosphorylation of CREB (cAMP response element-binding protein), a prominent downstream target of PKA, even with the concomitant application of the adenylate cyclase agonist forskolin to increase cAMP levels. Conversely, when cAMP levels were increased using the phosphodiesterase inhibitor IBMX, Rp-cAMPS potently inhibited CREB phosphorylation. Taken together, our results suggest that an alternate cAMP dependent pathway was involved in increasing CREB phosphorylation and neuroplasticity. This idea was supported by an in vitro neurite outgrowth assay, where inhibiting PKA did enhance neurite outgrowth. However, when PKA inhibition was combined with inhibition of EPAC2 (exchange protein directly activated by cAMP), another downstream target of cAMP in neurons, neurite outgrowth was significantly reduced. In conclusion, blocking PKA in cortical neurons of spinal cord injured rats increases neurite outgrowth of the lesioned corticospinal tract fibres and the efficacy of rehabilitative training, likely via EPAC.

A motorized pellet dispenser to deliver high intensity training of the single pellet reaching and grasping task in rats

The single pellet reaching and grasping (SPG) task is widely used to study forelimb motor performance in rodents and to provide rehabilitation after neurological disorders. Nonetheless, the time necessary to train animals precludes its use in settings where high-intensity training is required. In the current study, we developed a novel high-intensity training protocol for the SPG task based on a motorized pellet dispenser and a dual-window enclosure. We tested the protocol in naive adult rats and found 1) an increase in the intensity of training without increasing the task time and without affecting the overall performance of the animals, 2) a reduction in the variability within and between experiments in comparison to manual SPG training, and 3) a reduction in the time required to conduct experiments. In summary, we developed and tested a novel protocol for SPG training that provides higher-intensity training while reducing the variability of results observed with other protocols.