Peripheral Nerve Injury & Regeneration

• Peripheral nerve injury (PNI) often stems from accidents, war, or natural disasters, leading to motor and sensory neuron dysfunction
• This significantly diminishes patients' quality of life and creates a substantial societal burden
• In contrast to the central nervous system, the peripheral nervous system can regenerate spontaneously after injury
• This process requires intricate pathological and physiological changes, with cells and molecules cooperating to create a conducive microenvironment for regeneration and motor reinnervation

Microenvironment Dynamics

• The dynamic biochemical microenvironment is balanced by the extracellular matrix, blood and lymphatic vessels, and cell adhesion molecules
• Factors like cytokines and chemokines, secreted by Schwann cells, fibroblasts, endothelial cells, and immune cells, also play a role

Anatomical Structure

• Peripheral nerves comprise longitudinal axon bundles, myelinated glial cells (Schwann cells), and three-layered membranes (endoneurium, perineurium, epineurium)
• The endoneurium provides a matrix around axon units
• "Fascicles," or grouped nerve fibers, are covered by fibroblast-like cells inside the perineurium
• The epineurium wraps single or multi-fascicles, blood and lymphatic vessels, and adipose tissue and has inner and outer layers of collagen and connective tissue

Peripheral Nerve Injury (PNI) Types

• PNIs are classified into three types (neuropraxia, axonotmesis, and neurotmesis) by Seddon, and five types by Sunderland based on epineurium integrity
• Wallerian degeneration (WD) and Büngner band formation are crucial for nerve repair and regeneration

Wallerian Degeneration (WD)

• WD involves axon collapse, myelin breakdown, microtubule/neurofilament disarray, and cytoskeleton disassembly
• The proximal stump's axon sprouts a growth cone to explore the target tissue's microenvironment
• Schwann cells undergo dedifferentiation, proliferation, and migration after nerve injury, forming a bridge along the basement membrane called Büngner bands
• Reprogramming myelinating and non-myelinating Schwann cells is complex, and the repaired Schwann cells are critical for axonal regrowth and myelin debris clearance
• Schwann cells upregulate neurotrophic growth factors, express cytokines to recruit macrophages, and provide guidance cues for axon regeneration before re-myelinating axons and restoring nerve function

Innate Response

• Early research has found both neuroprotective and neurodestructive effects of immune responses, which are crucial for injured environment homeostasis

Inflammatory and Repair

• Functional recovery post-PNI involves inflammation and regeneration
• Cytokines, chemokines, secondary messengers, macrophages, neutrophils, natural killer cells, and T lymphocytes mediate inflammation
• Monocytes, neutrophils, and lymphocytes infiltrate the nerve lesion in a time-dependent manner
• The pro-inflammatory immune response remodels the distal stump microenvironment to disintegrate nerve fibers and phagocytize tissue debris
• Macrophages polarize to the M2 phenotype during the anti-inflammatory response, upregulating transcription factors to promote axon regeneration
• Natural killer cells and lymphocytes are reduced and T-cell activation is suppressed
• Failure to transition from pro- to anti-inflammatory response leads to chronic inflammation and neuropathic pain
• The immune response after PNI must be controlled to improve functional recovery
• Macrophages interact with Schwann cells, axons, neurons, fibroblasts, and endothelial cells and also release neurotrophic factors
• Other immune cells like neutrophils, NK, and T cells are essential for effective immune responses during peripheral repair and functional recovery
• How these immune cells interact with nerve repair and regeneration requires further study

Immune Targets

• Efforts to direct immune cell behavior or the immune microenvironment for nerve repair are being explored widely
• Strategies to regulate macrophages and T cells using tissue engineering, physical, and biochemical elements
• Recent data on immune responses during peripheral nerve repair and immune-cell-mediated engineering strategies provide a new insight for immunomodulatory therapies for peripheral nerve injury

Neutrophils

• Neutrophils are some of the first inflammatory cells to infiltrate the lesion site post-PNI
• Positive markers are detectable on days 3 and 9 post-injury in the injured sciatic nerves of mice, and Csf3r gene identifies mature neutrophils in the distal nerve stump
• Neutrophils are innate immune cells from bone marrow stem cells that last 24-48 hours, or longer during inflammatory reactions
• Roles consist of recruiting macrophages by secreting pro- and anti-inflammatory cytokines and chemokines to trigger initial inflammatory reactions
• Granules from neutrophils promote monocyte differentiation to macrophages, emphasizing neutrophils in functional macrophage polarization
• After sciatic nerve injury, myelin debris clearance is conducted by neutrophils
• Neutrophil depletion substantially inhibits myelin clearance in male wild-type mice and Ccr2¯/¯ mice
• Neutrophils accumulated at the epineurium in the WD area at 6 hours after injury, peaked at 12 hours, and disappeared by 1 day after injury
• Neutrophil extracellular traps (NETs) restricted macrophage infiltration into the parenchyma, influencing repair in WD
• Migration inhibitory factor MIF-CXCR4-NETs axis promotes repair in WD by reducing neutrophil accumulation
• Neutrophils cause hyperalgesia after nerve injury, and reducing these cells will reduce mechanical hyperalgesia in post-surgical pain
• Cytotoxicity in PNI has not been identified
• Crosstalk between neutrophils and macrophages can contribute to macrophage-mediated tissue repair

Macrophages and Monocytes

• Macrophages are mononuclear phagocytes in all tissues that act as phagocytic antigen-presenting cells
• They also maintain homeostasis and participate in multiple functional profiles
• There are two macrophage species in PNI
  • Tissue-resident macrophages from the yolk sac during embryogenesis
  • Monocyte-derived macrophages from hematopoietic stem cells in the bone marrow, which can be recruited to the injury site by cytokines
• Macrophages mediate tissue repair and provide a suitable microenvironment for regeneration in peripheral nerve repair
• Resident macrophages alongside Schwann cells or neutrophils respond to nerve injury first
• The recruitment of blood monocytes requires chemokines and proteins such as tumor necrosis factor α (TNFα), IL-1a, IL-1β, monocyte chemotactic protein 1 (MCP-1), leukemia inhibitory factor (LIF), and pancreatitis-associated protein III
• This event starts 2-3 days after injury and peaks at about 7 days
• Degenerated myelin removal by Schwann cells and macrophages can be independent
• Macrophages balance dynamic microenvironment in nerve lesion sites by participating in the phagocytosis of myelin debris and axon fragments
• They also drive PNR by simulating Schwann cells by supporting SC migration/maturation, ECM remodeling, promoting angiogenesis, preventing ectopic axon growth, and accelerating outgrowth

Macrophage Heterogeneity

• Macrophages in different tissues have specific subpopulations (e.g., Kupffer cells in the liver, splenic macrophages in the spleen, osteoclasts in the bone, and microglia in the brain)
• Heterogeneity may explain functional diversity
• Advances in single-cell transcriptomics can identify the specific cell subsets of tissues
• Recent study has found that harbored macrophages are distinct in the epineurium and endoneurium with specific spatial characterization in the PNS
• Subgroups of macrophages have differences in signature gene expression pattern, ranging from the PNS to the CNS, in response to the sciatic nerve crush injury
• Resident macrophages in the PNS recruit monocyte-derived macrophages after sciatic nerve injury and are mainly responsible for effective debris clearance
• Resident macrophages participate in axon regrowth, confirmed by the depletion of resident macrophages, which will lead to the complete failure of axon regeneration
• Macrophages can be divided into two classic groups
  • M1 macrophages are pro-inflammatory and are induced by lipopolysaccharides (LPS), toll-like receptor (TLR) ligands and interferon γ (IFN-γ), TNFα, and CCL2, which can aggravate inflammatory responses and the elimination of apoptotic cells and debris by secreting inflammatory factors

Macrophage Subtypes (M2)

• Further classified into four subtypes (M2a, M2b, M2c and M2d) based on differential activation pathways
• Subtypes are associated with anti-inflammatory response or pro-healing phenotype
  • M2a is induced by IL-4 and IL-13
  • M2b is induced by the immune complex
  • M2c is induced by IL-10 and transforming growth factor-beta (TGF-β)
  • M2d is induced by the A2AR agonist
• Subsets play anti-inflammatory roles through growth factors, removing apoptotic cells (M2a), promoting angiogenesis (M2b/d) and ECM synthesis (M2b/c)
• M1 and M2 macrophages are maintained in a dynamic balance within changes in microenvironment
• Delivery of IFN-y or IL-4 in a rat model confirmed prohealing M2a and M2c macrophages are in regenerative bias

Macrophage Plasticity

• It enables inflammatory and tissue damage
• Following PNI, M1 macrophages are dominant in WD and increase within 2 days post-injury
• M2 macrophages have a prominent action in the subsequent anti-inflammatory response and gradually replace the M1 phenotype from 3-7 days
• The different techniques identify marker genes of M1 or M2 have different results
• Macrophage-associated miRNAs were screened and up-regulated miRNAs (miR-18a, miR-19b, miR-21, miR-29) may regulate microenvironment remodeling after PNI
• Strategies always attribute the macrophage fate to the M2 phenotype. The role of M1 subsets should not be ignored
• Particularly, elucidating the equilibrium relationship of M1 and M2 in PNR will be valuable for nerve regeneration in future investigations

Natural Killers

• NK cells are innate lymphoid cells that lack antigen-specific receptors and are crucial in inflammation and adaptive immunity, producing a much faster immune reaction
• They play roles in both tumor immune supervision and induction of cascades of immune reaction synchrony with other immune cells
• NK cell-activating receptors mainly contain killer cell immunoglobin receptors (KIR-2DS, KIR-3DS), the NK group 2D (NKG2D, NKG2C), natural cytotoxic receptor (NCR), 2B4, and CD226; inhibitory receptors mainly contain KIR-2DL, killer cell lectin-like receptor (KLR, CD49/NKG2), and T cell immunoreceptor with Ig and ITIM domains (TIGIT)
• Cytokines (IL-12, TNF-a, IFN, IL-18) are secreted by macro-phages to stimulate NK cells and promote M2 macrophages polarized or switched to the M1 phenotype, which increases NK cell activation
• Cytotoxic NK cells infiltrate the lesion site within days of PNI and respond to retinoic acid early inducible protein 1 (RAE1), which is the NK group 2D (NKG2D) ligand expressed by injured sensory axons
• NK cell interactions complement WD and accelerate damaged axon clearance to accomplish the function recovery of the PNS
• Their immune-modulating role should also be investigated

T Cells

• T lymphocytes are adaptive immune cells that infiltrate the lesion site post-PNI, to peak level in 14-28 days
• T- and B-lymphocytes are absent in recombination activating gene 2 (RAG2¯/¯) mice
• Lymphocytes injected at the injury site in the acute phase of WD can improve nerve regeneration and sensory recovery
• Lymphocyte therapy can show positive effects on regenerative processes via improving debris clearance in the early phase of sciatic nerve injury
• T cells also participated in the neuropathic pain following the nerve injury
• The exact functions of different T cell subsets that accumulate at injury sites and the role of T cells in PNR has yet to be identified

T Cell Types

• Classified into CD4+ and CD8+ T cells
• TCD4+ T cells subpopulation contains
• T-helper cells (Th1, Th2 and Th17)
• Regulatory T cells (Treg), and these cells are essential for maintaining homeostasis after nerve injury
• The Th1-mediated immune response activates macrophages polarized into the M1 phenotype by secreting IFN-γ, and improve the neurotrophic factor expression and promote cell proliferation in SCs
• Th2 cells release IL-4, IL-5, and IL-13 and regulate macrophages toward the M2 phenotype to form an anti-inflammatory environment
• Bombeiro et al. used lymphocyte therapy to improve neve regeneration in traumatic injuries and revealed
• Th1 boost the inflammatory response during WD to improve the conduct microenvironment for axon recovery
• Compared with the control group, the recipient higher expressions of brain-derived neurotrophic (BDNF) at 7 days and 21 days
• Treg cells contribute to maintaining immune homeostasis and regulating the inflammatory response by suppressing the activation of other immune cells, is an intrinsic mechanism in pain

Regenerative Strategies

• Include immunosuppressive drugs, cytokine therapy, and chemokine therap

Repair

• Clinical peripheral nerve injuries (crushing, transection, stretching, neurological tumors, and combined damage) require end-to-end epineural tension-free suturing after surgery
• Nerve grafts (autografts or allografts), nerve transfer (bridge the proximal nerve to target the motor endplate to accomplish earlier reinnervation) and nerve conduits can be introduced, they have some disadvantages
• Tissue-engineered nerve grafts provide an altenratice strategy for neve repairment
• Nerve regeneration can be improved by recently developed artificial nerve conduits to improve the microenvironment
• Nerve regeneration is aided through scaffold-based materials combined with
• Physical properties (surface topography, fibers/hydrogel filled in conduits lumen structure)
• Chemical material composition, surface functional groups

Macrophages and Nerves

• Small pores, low roughness, and high substrate stiffness of biomaterials can stimulate macrophages into pro-inflammatory M1 subtypes
• Scaffolds that have a smooth shape, low stiffness, high roughness and hydrophilicity trigger macrophages to polarize into anti-inflammatory M2 phenotypes
• Micro patterns, stiffer substrates, and precise conductivity of NGCs can induce macrophage M2 polarization
• The materials that can produce such changes include, Chitosan, Hyaluronic acid (HA), alginate, and heparin

Macrophage Phenotypes in Nerve Repair

• A better repair was demonstrated in aligned fibers and the multifunctional biodegradable conductive hydrogel
• Better macrophage balance and functional recovery was shown among – M1 and M2 types – Stem cells and ECM