Peripheral nerve injuries induce a pronounced immune reaction within the spinal cord, largely governed by microglia activation in both the dorsal and ventral horns. The mechanisms of activation and response of microglia are diverse depending on the location within the spinal cord, type, severity, and proximity of injury, as well as the age and species of the organism. Thanks to recent advancements in neuro-immune research techniques, such as single-cell transcriptomics, novel genetic mouse models, and live imaging, a vast amount of literature has come to light regarding the mechanisms of microglial activation and alluding to the function of microgliosis around injured motoneurons and sensory afferents. Herein, we provide a comparative analysis of the dorsal and ventral horns in relation to mechanisms of microglia activation (CSF1, DAP12, CCR2, Fractalkine signaling, Toll-like receptors, and purinergic signaling), and functionality in neuroprotection, degeneration, regeneration, synaptic plasticity, and spinal circuit reorganization following peripheral nerve injury. This review aims to shed new light on unsettled controversies regarding the diversity of spinal microglial-neuronal interactions following injury. 

/pdf/the-role-of-microglia-in-neuroinflammation-of-the-spinal-225bvfg0.pdf

The Role of Microglia in Neuroinflammation of the Spinal Cord after Peripheral Nerve Injury

Abstract The prevalence and severity of many chronic pain syndromes differ across sex, and recent studies have identified differences in immune signalling within spinal nociceptive circuits as a potential mediator. Although it has been proposed that sex-specific pain mechanisms converge once they reach neurons within the superficial dorsal horn, direct investigations using rodent and human preclinical pain models have been lacking. Here, we discovered that in the Freund’s adjuvant in vivo model of inflammatory pain, where both male and female rats display tactile allodynia, a pathological coupling between KCC2-dependent disinhibition and N-methyl-D-aspartate receptor (NMDAR) potentiation within superficial dorsal horn neurons was observed in male but not female rats. Unlike males, the neuroimmune mediator brain-derived neurotrophic factor (BDNF) failed to downregulate inhibitory signalling elements (KCC2 and STEP61) and upregulate excitatory elements (pFyn, GluN2B and pGluN2B) in female rats, resulting in no effect of ex vivo brain-derived neurotrophic factor on synaptic NMDAR responses in female lamina I neurons. Importantly, this sex difference in spinal pain processing was conserved from rodents to humans. As in rodents, ex vivo spinal treatment with BDNF downregulated markers of disinhibition and upregulated markers of facilitated excitation in superficial dorsal horn neurons from male but not female human organ donors. Ovariectomy in female rats recapitulated the male pathological pain neuronal phenotype, with BDNF driving a coupling between disinhibition and NMDAR potentiation in adult lamina I neurons following the prepubescent elimination of sex hormones in females. This discovery of sexual dimorphism in a central neuronal mechanism of chronic pain across species provides a foundational step towards a better understanding and treatment for pain in both sexes. 

/pdf/sexual-dimorphism-in-a-neuronal-mechanism-of-spinal-3d2l3u8o.pdf

Sexual dimorphism in a neuronal mechanism of spinal hyperexcitability across rodent and human models of pathological pain

A critical-size bone defect is a challenging clinical problem in which a gap between bone ends will not heal and will become a nonunion. The current treatment is to harvest and transplant an autologous bone graft to facilitate bone bridging. To develop less invasive but equally effective treatment options, one needs to first have a comprehensive understanding of the bone healing process. Therefore, it is imperative to leverage the most advanced technologies to elucidate the fundamental concepts of the bone healing process and develop innovative therapeutic strategies to bridge the nonunion gap. In this review, we first discuss the current animal models to study critical-size bone defects. Then, we focus on four novel analytic techniques and discuss their strengths and limitations. These four technologies are mass cytometry (CyTOF) for enhanced cellular analysis, imaging mass cytometry (IMC) for enhanced tissue special imaging, single-cell RNA sequencing (scRNA-seq) for detailed transcriptome analysis, and Luminex assays for comprehensive protein secretome analysis. With this new understanding of the healing of critical-size bone defects, novel methods of diagnosis and treatment will emerge.

https://www.mdpi.com/2306-5354/9/4/171/pdf?version=1649815608

Novel Techniques and Future Perspective for Investigating Critical-Size Bone Defects

Background
Dysregulated inflammation is associated with many skeletal diseases and disorders, such as osteolysis, non-union of fractures, osteonecrosis, osteoarthritis and orthopaedic infections. We previously showed that continuous infusion of lipopolysaccharide (LPS) contaminated polyethylene particles (cPE) caused prolonged inflammation and impaired bone formation. However, the metabolic and bioenergetic processes associated with inflammation of bone are unknown. Mitochondria are highly dynamic organelles that modulate cell metabolism and orchestrate the inflammatory responses that involve both resident and recruited cells. Glycolytic reprogramming, the shift from oxidative phosphorylation (OXPHOS) to glycolysis causes inappropriate cell activation and function, resulting in dysfunctional cellular metabolism. We hypothesized that impaired immunoregulation and bone regeneration from inflammatory states are associated with glycolytic reprogramming and mitochondrial dysfunction in macrophages (Mφ) and mesenchymal stromal cells (MSCs).


Methods
We used the Seahorse XF96 analyzer and real-time qPCR to study the bioenergetics of Mφ and MSCs exposed to cPE. To understand the oxygen consumption rate (OCR), we used Seahorse XF Cell Mito Stress Test Kit with Seahorse XF96 analyzer. Similarly, Seahorse XF Glycolytic Rate Assay Kit was used to detect the extracellular acidification rate (ECAR) and Seahorse XF Real-Time ATP Rate Assay kit was used to detect the real-time ATP production rates from OXPHOS and glycolysis. Real-time qPCR was performed to analyze the gene expression of key enzymes in glycolysis and mitochondrial biogenesis. We further detected the gene expression of proinflammatory cytokines in Mφ and genes related to cell differentiation in MSC during the challenge of cPE.


Results
Our results demonstrated that the oxidative phosphorylation of Mφ exposed to cPE was significantly decreased when compared with the control group. We found reduced basal, maximal and ATP-production coupled respiration rates, and decreased proton leak in Mφ during challenge with cPE. Meanwhile, Mφ showed increased basal glycolysis and proton efflux rates (PER) when exposed to cPE. The percentage (%) of PER from glycolysis was higher in Mφ exposed to cPE, indicating that the contribution of the glycolytic pathway to total extracellular acidification was elevated during the challenge of cPE. In line with the results of OCR and ECAR, we found Mφ during cPE challenge showed higher glycolytic ATP (glycoATP) production rates and lower mitochondrial ATP (mitoATP) production rates which is mainly from OXPHOS. Interestingly, MSCs showed enhanced glycolysis during challenge with cPE, but no significant changes in oxygen consumption rates (OCR). In accordance, seahorse assay of real-time ATP revealed glycoATP rates were elevated while mitoATP rates showed no significant differences in MSC during challenge with cPE. Furthermore, Mφ and MSCs exposed to cPE showed upregulated gene expression levels of glycolytic regulators and Mφ exposed to cPE expressed higher levels of pro-inflammatory cytokines.


Conclusion
This study demonstrated the dysfunctional bioenergetic activity of bone marrow-derived Mφ and MSCs exposed to cPE, which could impair the immunoregulatory properties of cells in the bone niche. The underlying molecular defect related to disordered mitochondrial function could represent a potential therapeutic target during the resolution of inflammation.

Glycolytic reprogramming in macrophages and MSCs during inflammation.

The introduction of sex-as-a-biological-variable policies at funding agencies around the world has led to an explosion of very recent observations of sex differences in the biology underlying pain. This review considers evidence of sexually dimorphic mechanisms mediating pain hypersensitivity, derived from modern assays of persistent pain in rodent animal models. Three well-studied findings are described in detail: the male-specific role of spinal cord microglia, the female-specific role of calcitonin gene-related peptide (CGRP), and the female-specific role of prolactin and its receptor. Other findings of sex-specific molecular involvement in pain are subjected to pathway analyses and reveal at least one novel hypothesis: that females may preferentially use Th1 and males Th2 T cell activity to mediate chronic pain. 

Sex differences in mechanisms of pain hypersensitivity

Complex regional pain syndrome (CRPS) is a chronic pain disorder with a clear acute-to-chronic transition. Preclinical studies demonstrate that toll-like receptor 4 (TLR4), expressed by myeloid-lineage cells, astrocytes, and neurons, mediates a sex-dependent transition to chronic pain; however, evidence is lacking on which exact TLR4-expressing cells are responsible. We used complementary pharmacologic and transgenic approaches in mice to more specifically manipulate myeloid-lineage TLR4 and outline its contribution to the transition from acute-to-chronic CRPS based on three key variables: location (peripheral vs central), timing (prevention vs treatment), and sex (male vs female). We demonstrate that systemic TLR4 antagonism is more effective at improving chronic allodynia trajectory when administered at the time of injury (early) in the tibial fracture model of CRPS in both sexes. In order to clarify the contribution of myeloid-lineage cells peripherally (macrophages) or centrally (microglia), we rigorously characterize a novel spatiotemporal transgenic mouse line, Cx3CR1-CreERT2-eYFP;TLR4fl/fl (TLR4 cKO) to specifically knock out TLR4 only in microglia and no other myeloid-lineage cells. Using this transgenic mouse, we find that early TLR4 cKO results in profound improvement in chronic, but not acute, allodynia in males, with a significant but less robust effect in females. In contrast, late TLR4 cKO results in partial improvement in allodynia in both sexes, suggesting that downstream cellular or molecular TLR4-independent events may have already been triggered. Overall, we find that the contribution of TLR4 is time- and microglia-dependent in both sexes; however, females also rely on peripheral myeloid-lineage (or other TLR4 expressing) cells to trigger chronic pain.SIGNIFICANCE STATEMENT The contribution of myeloid cell TLR4 to sex-specific pain progression remains controversial. We used complementary pharmacologic and transgenic approaches to specifically manipulate TLR4 based on three key variables: location (peripheral vs central), timing (prevention vs treatment), and sex (male vs female). We discovered that microglial TLR4 contributes to early pain progression in males, and to a lesser extent in females. We further found that maintenance of chronic pain likely occurs through myeloid TLR4-independent mechanisms in both sexes. Together, we define a more nuanced contribution of this receptor to the acute-to-chronic pain transition in a mouse model of complex regional pain syndrome.

https://www.jneurosci.org/content/jneuro/41/19/4349.full.pdf

Temporal Contribution of Myeloid-Lineage TLR4 to the Transition to Chronic Pain: A Focus on Sex Differences.

Orthopedic injury can occur from a variety of causes including motor vehicle collision, battlefield injuries or even falls from standing. Persistent limb pain is common after orthopedic injury or surgery and presents a unique challenge, as the initiating event may result in polytrauma to bone, muscle, and peripheral nerves. It is imperative that we understand the tissue-specific and multicellular response to this unique type of injury in order to best develop targeted treatments that improve healing and regeneration. In this Mini Review we will first discuss current rodent models of orthopedic trauma/complex orthotrauma. In the second section, we will focus on bone-specific outcomes including imaging modalities, biomechanical testing and immunostaining for markers of bone healing/turnover. In the third section, we will discuss muscle-related pathology including outcome measures of fibrosis, muscle regeneration and tensile strength measurements. In the fourth section, we will discuss nervous system-related pathology including outcome measures of pain-like responses, both reflexive and non-reflexive. In all sections we will consider parallels between preclinical outcome measures and the functional and mechanistic findings of the human condition.

/pdf/modeling-complex-orthopedic-trauma-in-rodents-bone-muscle-4dv11jcyo1.pdf

Modeling Complex Orthopedic Trauma in Rodents: Bone, Muscle and Nerve Injury and Healing.

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Temporal Contribution of Myeloid-Lineage TLR4 to the Transition to Chronic Pain: A Focus on Sex Differences.

Modeling Complex Orthopedic Trauma in Rodents: Bone, Muscle and Nerve Injury and Healing.