Peripheral Nerve Regeneration

Objectives

  • Discuss the basics of regeneration

  • Examine peripheral nerve anatomy

  • Identify types of axonal nerve injury

  • Explore peripheral nerve degeneration

  • Understand peripheral nerve regeneration

  • Compare PNS regeneration to CNS regeneration

Regeneration Basics

Overview of Regeneration

  • All organisms possess the capability to regenerate; however, the degree of regeneration varies significantly across species and level of organizational complexity.

    • Planaria (Flatworm): Can regenerate an entire organism from a single piece of its body.

    • Carrot Cell: A single carrot cell can regenerate into an entire plant.

    • Liver in Vertebrates: Among vertebrates, the liver shows relatively better regenerative capability.

    • General Trend: Vertebrates, in comparison to invertebrates, tend to have a lower capacity for regeneration.

Mechanisms of Regeneration

Regenerative mechanisms can be broadly categorized based on:

  1. Origin of Regenerating Cells: This determines whether new cells arise from the same type of cells or different types.

  2. Characteristics of Regenerative Cells: These can be fully differentiated cells or stem cells.

  3. Activities of Regenerative Cells: Understanding if cells de-differentiate before re-differentiating.

  4. Regulatory Factors: Varied factors play roles in multiple regenerative activities; for instance:

    • Transforming Growth Factor-Beta (TGF-β): Involved in epithelial to mesenchymal transitions.

    • Fibroblast Growth Factor 2 (FGF2): Often facilitates de-differentiation.

    • Regeneration of various tissues could involve a mixture of these mechanisms.

Specific Methods of Regeneration

  1. Cellular Re-growth:

    • Mechanism involves the loss of the cytoplasmic portion of the cell; regrowth happens from the remaining part of the cell.

    • Illustratively, Schwann cells, which encase the nerve fibers, become essential for guiding the regenerative processes.

  2. Regeneration from Pre-existing Parent Cells:

    • Compensatory Hyperplasia: Cells proliferate while continuing to perform their usual functions.

      • Example: Liver hepatocytes and B cells of pancreatic islets undergo proliferation while maintaining differentiated structures.

    • De-differentiation and Re-differentiation: Cells revert to a less differentiated state to allow proliferation, as seen in limb regeneration in urodele amphibians.

      • Involves epithelial to mesenchymal transformation (EMT) and its reverse, MET.

  3. Transdifferentiation:

    • Direct conversion of one cell type into another; two pathways:

      • Direct: New gene expression occurs while repressing previous patterns.

      • Indirect: De-differentiation into a progenitor cell which then differentiates into a new type.

  4. Activation of Adult Stem Cells:

    • Adult stem cells divide asymmetrically, yielding one stem cell and one lineage-committed daughter cell.

    • They proliferate to generate specific precursor cells that will eventually form terminal cell types.

    • Activation triggers can vary depending on tissue type (injury triggers rapid division, while maintenance involves slow continuous division).

Peripheral Nerve Anatomy

  • Structure: A peripheral nerve consists of numerous axons along with connective tissue, blood vessels, and adipose tissue.

    • Axons are organized into bundles known as fascicles.

    • Endoneurium:

      Delicate tissue that wraps around the myelin sheath of each myelinated fiber (Latin: "within").

    • Perineurium: A connective tissue sheath encasing a fascicle (Latin: "surrounding").

    • Epineurium: The outermost layer of connective tissue enveloping a peripheral nerve (Latin: "outer").

Myelin Sheath

  • In the peripheral nervous system (PNS), myelin sheaths are produced by Schwann cells.

  • Neurolemma: Encompasses only the axon (excluding dendrites or soma) and acts as an insulator by preventing ion dissipation during action potentials.

  • Node of Ranvier: Gaps in Schwann cell coverage where voltage-gated $Na^+$ and $K^+$ channels are located, allowing propagation of action potentials.

≥≥Schwann Cells

  • Role: Each myelinating Schwann cell exclusively myelinates a single axon. The outermost layer of the neurolemma is composed of the cytoplasm and nucleus of the Schwann cell.

  • Guiding Regeneration: Schwann cells serve as tubes that direct regenerating axons and will not myelinate thin fibers – multiple fibers may correspond to a single Schwann cell.

  • Origin: Schwann cells originate from neural crest cells and are crucial for nerve development.

  • Associative Behavior: They form myelin accordingly based on the size of the associated axon, with larger fibers receiving myelination through radial sorting, while those associated with smaller fibers may remain non-myelinating.

  • Specialized Types: Some Schwann cells associate with synapses, while others form encapsulating end organs such as the Pacinian corpuscle.

  • Dependency of Schwann Cell Precursors and Neurons

    • Schwann cell precursors provide trophic support to ensure neurons receive the necessary sustenance prior to targeting innervation.

  • There exists a mutual dependency where Schwann cell precursors rely on neurons for guidance in matching the number of nerves with Schwann cells.

Peripheral Nerve Damage

Types of Nerve Damage - Seddon's Classification

  1. Neuropraxia (low severity): Structure of nerve remains intact, but electrical conduction is impaired. Full recovery usually occurs within hours to weeks. Example: Ischemia or compression injury.

  2. Axonotmesis: Disruption of the axon occurs while the myelin sheath remains intact. Recovery via regeneration is possible, taking from weeks to years depending on damage severity. Example: Crush injury.

  3. Neurotmesis: Involves damage to the nerve trunk alongside surrounding tissue, with severe cases necessitating complete transection. Recovery is uncertain and can result in cell death if the soma is affected.

Sunderland's Classification of Nerve Damage

  • This classification provides further detail on degrees of injury, from 1st to 5th:

    • 1st Degree: Equivalent to neuropraxia.

    • 2nd Degree: Equivalent to axonotmesis.

    • 3rd Degree: Axonotmesis with endoneurium disruption.

    • 4th Degree: Axonotmesis with endoneurium and perineurium disruption.

    • 5th Degree: Equivalent to neurotmesis (complete transection).

Peripheral Nerve Degeneration

Types of Degeneration

  1. Wallerian Degeneration: Occurs when both axons and surrounding tissues are damaged (as seen in neurotmesis).

  2. Demyelination: This primarily affects Schwann cells; axons may be secondarily impacted and is typically observed in neuropraxia.

  3. Axonal Degeneration: Here, the axons are directly damaged while the Schwann cells remain (as seen in axonotmesis).

Wallerian Degeneration Described

  • Involves extensive nerve damage leading to myelin degeneration, loss of endoneurium, and perineurium.

  • Develops within 7-10 days post-injury: Stumps swell, retract, and distal axons disintegrate. Microglia and macrophages infiltrate damaged areas, clearing debris and initiating repair.

    • The nerve cell nucleus shifts eccentrically, with Nissl bodies disappearing (chromatolysis).

    • Retracted presynaptic terminals lead to both retrograde and anterograde degeneration of neurons.

  • This phase is crucial for preparing the environment for potential regeneration.

Demyelination

  • Primary lesion affects Schwann cells without immediate axonal damage, leading to slowed conduction or conduction block distal to the lesion, exemplified in conditions like Guillain-Barre syndrome and certain neuropathies (e.g., Charcot-Marie-Tooth).

Peripheral Nerve Regeneration

  • Mechanisms of Regeneration After Nerve Transection

    • Nerve Bridge Formation: After proximal and distal stumps retract, they reconnect via a bridge composed of extracellular matrix and inflammatory cells (macrophages, neutrophils, and fibroblasts).

  • Initially poorly vascularized, these areas exhibit a lack of oxygen, leading macrophages to express vascular endothelial growth factor (VEGF), promoting angiogenesis.

  • Role of Macrophages in Regeneration

    • Macrophages release inflammatory components that mobilize the immune response (e.g., prostaglandins, IL1, IL6, IL18, TNF, LIF).

  • Within ganglia, they are recruited by CCL2 and increase the expression of regeneration-associated genes (RAGs).

  • As monocyte-derived macrophages migrate into the nerve bridge, they release VEGF, enhancing endothelial migration and proliferation.

  • In the distal stump, macrophages aid Schwann cells in creating a favorable environment for axonal regrowth by clearing away any debris.

Role of Schwann Cells in Regeneration

  • Following injury, Schwann cells sense damage and undergo de-differentiation into a progenitor state while secreting attractants for monocyte macrophages.

  • They assist in debris clearance while remodelling the extracellular environment and migrate along regrowing vasculature to form Bands of Bungner to guide regrowing axons in the distal stump.

Regrowth of Nerve Axons

  • The proximal nerve stump generates multiple collateral branches that may reach the nerve bridge and distal tube via the Bands of Bungner. Successful regeneration to target tissues may take weeks or months, depending on various factors, including age.

  • Target muscles can atrophy without innervation but can regain mass upon reinnervation. Notably, postsynaptic neurons may face cell death due to transsynaptic effects if reinnervation is delayed.

    • Reference: Arslantunali et al., 2014.

Peripheral Nerve Regeneration at the Neuromuscular Junction (NMJ)

  • Components of NMJ: The NMJ includes presynaptic synaptic boutons and a specialized extracellular matrix enriched with proteins influencing synaptic development and maintenance (e.g., S-laminin, agrin, rapsyn).

  • During degeneration, although nerve fibers and synapses deteriorate, junctional complexes remain intact, allowing for post-injury biological processes.

  • Following injury, if the endoneurium and perineurium aren't damaged, axonal regeneration may ensue rapidly.

  • Schwann cells and macrophages play critical roles in nerve cleanup and regenerative activities without the need for bridging.

  • Summary of Peripheral Nerve Regeneration

    • Regeneration can occur via four mechanisms: cellular regrowth, regeneration via pre-existing cells, activation of adult stem cells, and transdifferentiation.

  • Anatomy involves axons ensheathed by Schwann cells mixed with connective tissues: the endoneurium, perineurium, and epineurium.

  • Schwann cell types influence whether myelination occurs; degeneration pathways follow Wallerian degeneration, axonal, or demyelination routes.

  • Regeneration includes debris clearance, nerve bridge formation, Schwann cell involvement, and reconnection with targets.

Differences Between PNS and CNS Regeneration

Environmental Changes Post-Development

  • Post-development, environmental guidance cues diminish while differentiated cell types—not present earlier—now express inhibitory molecules or undergo changes due to injury.

  • Immune system maturation effects additionally complicate CNS regeneration.

  • Myelin-Associated Inhibitors in CNS

    • CNS regeneration is impeded by myelin-associated inhibitors like myelin-associated glycoprotein (MAG), NoGoA, oligodendrocyte myelin glycoprotein (Omgp), Ephrin B3, and semaphorin 4D.

  • These molecules activate RhoA signaling in axonal growth cones, potentially leading to growth cone collapse.

    • Reference for these findings: Yiu and He, 2006.

Hostile Environment in CNS

  • CNS regeneration is further inhibited by a reactive environment generated by myelin-associated inhibitors that inhibit regrowth and by recruitment and inflammatory properties of reactive astrocytes leading to glial scar formation.

  • Glial scars, often co-occurring with fluid-filled cysts, exacerbate the physical and biochemical barriers to axonal regrowth.

Reactive Astrocytes Dynamics

  • Astrocytes perform many supportive roles in the CNS, but upon injury, they can undergo reactive gliosis—a response to inflammatory signals characterized by hypertrophy and de-differentiation. Some proliferate, contributing to chronic inflammation and glial scar formation that inhibits regrowth.

    • Reference: Yiu and He, 2006.

Intrinsic Neuronal Differences

  • Differences also exist intrinsically among embryonic neurons, PNS neurons, and CNS neurons, affecting regenerative capability (Mar et al., 2014).

Summary: PNS versus CNS Regeneration

  • The glial cells in the CNS encourage regeneration-inhibitory environmental changes. Oligodendrocytes and myelin debris express molecules that prevent regrowth, while astrocytes facilitate scar formation that poses biophysical challenges.

  • Intrinsic changes in CNS neurons limit regenerative potential, reflected in the absence of increased histone acetylation or regeneration-associated gene expression, decreased calcium and protein synthesis, along with the presence of regeneration inhibitors such as SOCS3, pTEN, and EFA-6.