Transcriptional reprogramming post-peripheral nerve injury: A systematic review

Neuropathic pain is characterised by periodic or continuous hyperalgesia, numbness, or allodynia, and results from insults to the somatosensory nervous system. Peripheral nerve injury induces transcriptional reprogramming in peripheral sensory neurons, contributing to increased spinal nociceptive input and the development of neuropathic pain. Effective treatment for neuropathic pain remains an unmet medical need as current therapeutics offer limited effectiveness and have undesirable effects. Understanding transcriptional changes in peripheral nerve injury-induced neuropathy might offer a path for novel analgesics. Our literature search identified 65 papers exploring transcriptomic changes post-peripheral nerve injury, many of which were conducted in animal models. We scrutinize their transcriptional changes data and conduct gene ontology enrichment analysis to reveal their common functional profile. Focusing on genes involved in 'sensory perception of pain' (GO:0019233), we identified transcriptional changes for different ion channels, receptors, and neurotransmitters, shedding light on its role in nociception. Examining peripheral sensory neurons subtype-specific transcriptional reprograming and regeneration-associated genes, we delved into downstream regulation of hypersensitivity. Identifying the temporal program of transcription regulatory mechanisms might help develop better therapeutics to target them effectively and selectively, thus preventing the development of neuropathic pain without affecting other physiological functions.


Systematic search, screening, and inclusion/exclusion criteria
A literature search was conducted across four databases: Medline, Embase, Scopus and Web of Science (Supplementary Figure 1, Table 1).Inclusion criteria were primary research articles that investigated (i) transcriptional changes (ii) in PSNs (iii) following PNI.
Nonessential words were removed from the research question to define the search terms 'peripheral nerve injury', 'transcriptional reprogramming', and 'neuropathic pain'.Truncation and wildcards were applied to all synonyms to include all relevant papers; synonyms were pooled with 'OR'.'AND' was used to combine search terms.Abstract screens were conducted to exclude papers not relevant to our research question.Publications not in English were also excluded.A full-text screen was then conducted to categorise studies by the methods used to analyse transcriptional changes and cell types analysed.Papers that did not identify transcriptional changes in PSNs were excluded.The snowball sampling method was used to identify additional seminal papers from in-paper references found during full text screen.

Data management and analysis
A collective Gene Ontology (GO) enrichment analysis was conducted on the studies that utilised single cell or single nucleus RNA sequencing (scRNA-seq and snRNA-seq, respectively) for genome-wide transcriptomic profiling to determine the common functional profile of PNI-induced TR of PSNs.Genes identified as upregulated or downregulated following PNI were collated, and analysis was performed using Metascape [24][25][26] .Further

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Description of the included studies
We have identified a total of 65 primary research articles that analysed transcriptional changes in PSNs following PNI 10,13,23, . Only our studies reported TR in humans with preexisting peripheral neuropathy 744,63,65,88 .Transcriptomic analysis studies on human sensory neurones in the context of PNP used nerve biopsy or post-mortem samples from patients with diagnoses of peripheral neuropathy (either chemotherapy-induced, traumatic, or painful diabetic neuropathy).However, study populations were often small and heterogenous, a study reported only male subjects (n = 10) 63 , two studies included males and females (69 males and 19 females 65 , age 62; and 6 males and 6 females, age 56.6-45.6 88 ), and one that did not specify gender 44 (n = 50; breast cancer survivor however point at females).
The most common methods used to analyse transcriptional changes were RNA-sequencing (n = 39) and microarray analysis (n = 13).RNA-sequencing uses high-throughput sequencing to determine expression levels of all genes within a sample 89 , while microarrays can be used to measure expression levels of predetermined genes or compare gene expression between cells 90 .Other techniques included immunohistochemistry, GeneChip Arrays, and quantitative polymerase chain reaction (qPCR), including real-time (RT-PCR) 65 .
It is generally viewed that TR-induced changes in protein expression result in increased neuronal excitability, enhanced spinal nociceptive input and, ultimately, PNP 1,12,22 .Given the potent ability of scRNA-seq and snRNA-seq to identify cell-specific transcriptional changes, alongside the use of animal models in studying molecular mechanisms underlying PNP, for

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Journal Pre-proof the remainder of this review focuses on studies that utilised these methodologies to analyse PNI-induced TR in PSNs.

Transcriptional Reprogramming in Primary Sensory Neurons following
Peripheral Nerve Injury scRNA-seq/snRNA-seq approaches gather gene expression data at individual cells.The expression profiles of specific cells are referred to as their cellular signature.In peripheral neuropathy, this is particularly important as sensory nerve fibres running in peripheral nerves originate from a heterologous population of neurons 91,92 .Several attempts have been made to classify PSNs into subtypes based on their cellular signatures in physiological conditions 91,93,94 .Although several sub-groups may be present within the PSN subtypes defined by various studies, generally five major categories can be identified based on differential sensory modality/functions and expression profiles (Table 4) 10,72 .Within the largediameter myelinated PSNs, two major categories were identified: proprioceptors and lowthreshold mechanoreceptors (LTMRs), both of which expressed NF200, Nefh.Regarding small diameter PSN's, the non-peptidergic (NP) and peptidergic (PEP) PSN subtypes had overlapping sensory modalities of nociception and thermoception, while the NP subtype also encompassed chemoreception and pruriception (itching).A further small diameter PSN subtype, c-LTMRs, were classified based on their expression pattern of Nefh-, Th+, Fam19a4+ (Table 4).Moreover, while some PNI-induced gene expression changes are common across all subtypes, others have been shown to differ between subtypes 72,77 .Six primary studies have utilised scRNA-seq for PSN-specific genome-wide transcriptomic profiling in rodent models of PNI 10,30,68,72,73,77 .Our GO enrichment analysis of these studies identified thirty-four genes as upregulated following PNI, with twenty varying functions, including, three that encode neurotransmitters, such as Tac1, Tac2, and NPY, which transcribe as peptides substance P (SP) and neurokinin A (NkA), neurokinin B (NkB) and neuropeptide Y, respectively; and three genes codifying transcription factors, Sox11, Stat3 and KLF6.Additionally, the expression of three transmembrane proteins has been found increased: Ankyrin-3 (encoded by the gene Ank3), also known as ankyrin-G and with a role in the formation and maintenance of the axon initial segment which also seems to contribute to spontaneous activity in neuropathic pain 95 ; fibronectin leucine rich transmembrane protein 3 (FLRT3), implicated in cell adhesion/receptor signalling; and the gene codifying for Semaphorin 6A (Sema6a) which has been previously associated with peripheral nerve injury-induced neuropathic pain 96 (Table 5).
Our GO enrichment analysis also identified twenty-seven downregulated genes including, among others: the voltage-gated sodium channels Scn9a, Scn10a and Scn11a which

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Journal Pre-proof respectively encode the alpha-subunits Na v 1.7, Na v 1.8 and Na v 1.9; different voltage-gated potassium channels of which stands Kcna2, the downregulation of which has been previously associated with the development of neuropathic pain 97 ; and the gene codifying for the glutamate ionotropic receptor AMPA type subunit 4 (Gria4), associated with neuronal excitation within different physiological and non-physiological processes.
To further assess the functional implications of this patterns of TR, we used Metascape to reveal the association of the differentially expressed genes with their overlapping functions related to nerve injury, regeneration, and pain.The most significant pathway-and processenriched cluster terms from the upregulated genes included regulation of signalling pathways and neuron projection development (Figure 2 [A], Figure 3 [A]), while those of the downregulated genes included sensory perception of pain and transmembrane transport (Figure 2 [A], Figure 3 [A]).
Furthermore, analysis using the DisGeNET database, which associates genes with disease processes, revealed that upregulated genes had the highest association with neuralgia and downregulated genes with hyperalgesia (Figure 4) 98 .

DISCUSSION
Given scRNA-seq and snRNA-seq's potent ability to identify cell-specific transcriptional changes, we focused our analysis on studies that employed these methods to analyse PNIinduced TR in PSNs.Due to limitations with human studies, analysis included only animal studies.This analysis, with particular focus on the functional classification of transcriptional changes in the context of PNP, revealed various components of the "emergency programs", as discussed below.

Neurons from the Mechanical Allodynia Associated Clusters (MAACs) play a major role in PNP
MAACs are a PNI-associated PSN subtype reported by Zhou and co-workers originating from peptidergic nociceptors following injury 77 .MAACs are of particular interest as they show downregulation of injury response genes, which tend to be upregulated in other neuronal subtypes following PNI, such as Adcyap1.The Adcyap1 gene encodes for pituitary adenyl cyclase-activating polypeptide, which is implicated in the induction of NP 99 .Additionally, MAAC gene expression is dominated by the Hobx7 gene, as opposed to genes encoding the TFs that dominate TR in other neuronal subtypes (e.g., activating transcription factor 3 -Atf3in large diameter myelinated -LM-, peptidergic and nonpeptidergic nociceptors) 10 .
Finally, genes most highly expressed in MAACs significantly map to various GO terms including "neurofilament bundle assembly" and "regulation of cell shape", thus suggesting J o u r n a l P r e -p r o o f Journal Pre-proof that cytoskeletal restructuring is associated with the development of mechanical allodynia 77 .
This finding is in opposition to the mechanisms underlying neuronal hyperexcitability reported in other clusters such as Large Diameter Myelinated (LM), peptidergic and nonpeptidergic neurons 77 .

Hypersensitivity
Voltage-gated ion channels and their relation to opioid receptors are an essential part of neuronal signalling, with their reprogramming forming a major component of PNI-induced pain.PNI-induced downregulation of opioid receptor mu 1 (Oprm1) 76 and opioid receptor kappa 1 (Oprk1) 77 has been reported in PSNs.These receptors, when activated by endogenous opioids or exogenous opioid medication, trigger signalling cascades that inhibit pain sensation 100 .They are involved in adenylyl cyclase inhibition and activation of the mitogen-activated protein kinase (MAPK) pathway to regulate nociception 100 .Activation of opioid receptors also inhibits voltage-gated calcium channels (VGCCs) and activates voltage gated potassium channels (VGKCs), preventing influx of calcium ions into neurons, and promoting efflux of potassium ions 100 .These changes result in highly negative membrane potentials, reducing the likelihood of reaching the threshold potential required for action potential generation hence, transmission of pain sensation.Downregulation of these antinociceptive mechanisms in injured neurons results in hyperexcitability and increased spinal nociceptive input that drives PNP.Interestingly, downregulation of Orpm1 can also be caused by morphine use, thus linking both opioid tolerance and NP in opioid users 101 .
This mechanism is augmented by downregulation of genes encoding VGKCs (e.g., Kcna2, Kcnb2, Kcng3, Kcnv1) 72,73,76 .Reduced potassium ion efflux from neurons leads to a resting membrane potential much closer to the threshold for action potential generation, thus increasing neuronal hyperexcitability 102 .Therefore, downregulation of the genes encoding VGKCs due to nerve injury may, partially, explain the NP-associated hypersensitivity.
Cacna2d1, a gene encoding a subunit of VGCCs, is also upregulated following PNI 72 .This potentiates NMDA receptor activity on both pre-and post-synaptic membranes, which causes hypersensitivity by reducing the magnitude of the stimulus required to surpass threshold potential 103 .Accordingly, an upregulation of Cacna2d1 has also been found in chronic NP associated with osteoarthritis 104 .

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Journal Pre-proof PNI-induced TR of several genes involved in detection of injurious stimuli has also been reported 10,72,77 .Errors in this mechanism and its resolution may lead to hypersensitivity and PNP.
Acid-sensing ion channels (ASICs) are involved in detecting acidotic conditions produced at the site of injury.Among them, stands ASIC1, encoded by the Asic1 gene, and a key component of this system 105 .ASICs are present in PSNs and act as proton sensors 106 .
Genes encoding ASICs are downregulated in MAACs post-PNI, increasing the threshold required to generate an action potential 77 .Conversely, upregulation of Asic1 has been reported in inflammatory models of PNI where acid hypersensitivity may result in PNP [105][106][107] .
Moreover, blocking ASICs has been shown to produce analgesia, suggesting that blocking the PNI-induced Asic1 upregulation may reduce PNP 108 .
P2rx3 encodes the P2X purinoceptor-3 (P2X3).Purinergic channels detect ATP released by damaged cells 61 .P2rx3 expression is mostly limited to nonpeptidergic PSNs and is upregulated specifically in nonpeptidergic neurons following injury 72 .Previous investigations identified P2rx3 as downregulated in peptidergic neurons 109 , however more recent evidence points to P2xr3 being expressed much more prominently in nonpeptidergic neurons 72 .
Increased P2X3 lowers the thresholds to generate action potentials, resulting in hypersensitivity.This may function as an adaptive mechanism to prevent further tissue damage following initial insult.However, failure to resolve this change following recovery may lead to persistent PNP 61 .Chronic NP is linked to increased P2rx3 expression in the context of dorsal root ganglion (DRG) nerve compression such as in disc herniation 110 .
Interestingly, P2rx3 expression levels also correlate with the perceived degree of pain experienced 111 .
Anoctamin-1 is upregulated in response to PNI 10 , potentially leading to mechanical and/or thermal hyperalgesia.Anoctamin-1 is a calcium-activated chloride ion channel that seems to influence both mechanical and thermal responses.Shear stress triggers membrane depolarisation via Anoctamin-1 and, interestingly, knocking out anoctamin-1 in mice results in reduced hyperalgesia in response to thermal damage 112,113 .Additionally, anoctamin-1 might also be involved in the increased release of calcium from intracellular stores 114 .

Neurotensin as a Key Neurotransmitter in PNP
The NTS gene, which encodes the neurotransmitter neurotensin, is upregulated in injured PSNs 76 .Neurotensin modulates pain responses and produces dose-dependent neuronal hypersensitivity by reducing the firing thresholds of the neurons 115 .Furthermore, NTS knockout in mice is linked to antinociceptive outcomes [116][117][118] .Accordingly, the upregulation in NTS induced by PNI could cause increased spinal nociceptive input and PNP.Moreover,

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Journal Pre-proof failure to return NTS expression to baseline levels following injury resolution may result in persistent PNP 115 .However, neurotensin's functions are not restricted to nociceptive signalling as it has also been associated with the control of blood pressure, substance abuse and several psychiatric disorders 119 .Therefore, blocking neurotensin as a therapeutic target for PNP might not be the best approach as it might have significant implications for other physiological processes.

Paradoxical Downregulation of Hypersensitivity Associated Genes
Genes Scn9a and Scn11a encode for two α subunits (Na V 1.7 and Na V 1.9, respectively), crucial parts of the voltage-gated sodium channels (VGNCs) and selectively expressed in PSNs from nociceptive pathways 120 .Upregulation of these genes may contribute to PNP through reduced threshold potentials and increased ectopic firing 120 .Moreover, Scn gain-offunction mutations have been identified within individuals with inherited painful conditions, while loss-of-function mutations are associated with reduced pain sensitivity 121 .However, studies utilising genome-wide profiling of injured PSNs have reported downregulation of both Scn9a and Scn11a 72,73,76 , while studies investigating the role of VGNCs in PNP demonstrated reduced expression in DRG neurons post-PNI 122,123 .This incongruency should be taken into consideration and urges to re-evaluate the role of VGNCs in PNP.
Similarly conflicting evidence can be seen for the transient receptor potential channels (TRPs).A great among of literature reports TRPs' dominant presence in PSNs with strong implications for hypersensitivity following tissue injury 124,125 .However, studies utilising sc/snRNA-seq to investigate PSN changes reported downregulation of both TRP Vanilloid Subfamily Member 1 (TRPV1) and TRP Ankyrin Subfamily Member 1 (TRPA1) following PNI 73,76,77 .Considering the potential clinical relevance of TRPs as therapeutic targets in PNP [126][127][128] , this peripheral downregulation should be the focus of further research.
Similarly, the Tac1 gene is downregulated in PSNs following PNI 68,76,77 .Tac1 encodes tachykinin neurotransmitters involved in pain perception via substance P, neurokinin A, neuropeptide K and neuropeptide Y, among others.Hence, downregulation of Tac1 could suggests that the neurons are failing to downregulate tachykinins post-PNI which may then contribute to hypersensitivity.However, this hypothesis is not yet supported by a comprehensive body of evidence.Additionally, conflicting data exists concerning tachykinins in PNP.While substance P antagonists showed inefficacy in pain 129 , studies involving Tac1 knockout mice revealed antinociceptive effects 130,131 .
Finally, an additionally seemingly paradoxical change is the upregulation of genes encoding tandem pore domain potassium ion channels (leak channels) such as Kcnk12, Kcnk13 and Knck16 which have been reported post-PNI 73 .These channels allow potassium ions to leak

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Journal Pre-proof out of neurons, hence increased expression hyperpolarises neurones and thus limits neuronal excitability 132 .However, it is important to note that changes in the expression of cationic channels should be examined in context of changes in anionic homeostasis, as the balance of these determine the excitability of the neurons 133 .
A great number of regeneration-associated genes (RAGs) are expressed in PSNs early in the time-course following PNI 10,30,68,72,73,76,77 .This expression pattern reflects the role of RAGs in facilitating early TR that aids functional recovery 134 .It is possible however, that RAGs may eventually modulate downstream expression of genes associated with hypersensitivity and allodynia 10 .
Renthal and colleagues reported one RAG, Atf3, to be essential for the post-injury cellular plasticity required for TR 10 .Atf3 has been shown to operate in tandem with c-Jun and Sprr1a, among other RAGs, in a multimodal transcriptional network the exact targets of which are yet not fully known 135,136 .Interestingly, Chang colleagues recently demonstrated that Atf3 acts as an upstream modulator of diabetic peripheral neuropathy (DPN) 137 while Kan and co-workers reported that Atf3 loss-of-function could prevent DPN 138 .
In addition to their upstream potential in TR-induced PNP, RAGs are also associated with neuron growth following injury 134 .Costigan et al. demonstrated that outgrowth of afferent nerve fibres in response to injury may further contribute to PNP through dorsal redistribution of low-threshold afferent nerves into lamina II of the spinal cord 12 .This indicates a link between PNI-induced TR and subsequent anatomical alterations that occur centrally, further up in the nociceptive pathway 139,140 .Therefore, given this interaction with the central nervous system (CNS), these changes are an important consideration for therapeutics.

Limitations
PNI-induced TR also alters gene expression in non-neuronal cells including Schwann cells, microglia, and fibroblasts and subsequent input from these cells may contribute to neuronal hyperexcitability 1,10,70 .Moreover, various central mechanisms have been implicated in sensory dysfunction following PNI 22 .Therefore, one limitation of this review is that we did not include TR that could take place post-PNI in non-neuronal or CNS-related tissues.However, even in the absence of mediation by non-neuronal cells and central mechanisms, direct PSN changes would likely still result in NP, as PNI-induced increases in spinal input from PSNs are an essential part of nociceptive processing in the CNS.However, further PNI-induced reprogramming is essential for the maintenance of this nociceptive processing.The studies analysed in this review are largely from animal models, therefore non-neuronal cell induced

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Journal Pre-proof transcriptional changes that reached statistical significance are evident in the scRNA-seq and snRNA-seq data.Finally, when considering therapeutic implications, targeting peripheral nociceptors has become the preferred approach owing to the promise of reduced undesirable CNS-mediated adverse effects 141 .
Studies investigating PNI-induced TR utilise various animal models of PNP to detect transcriptomic changes and gene regulation 10,30,68,72,73,76,77 .However, several limitations (Table 2 and Table 3) may hinder the translational validity of these models, including absence of inflammatory components and variability in neuronal damage, among others 142 .
Moreover, it is highly likely that TR differs between species (species-specific transcriptomic differences) 18 .Nonetheless, recent progress has begun addressing this limitation: by profiling molecular signatures from human organ donor nociceptors, researchers aim to correlate these signatures with those observed in animal models 36,143 .Another translational limitation is in regards of their temporality of TR as the transcriptome analysis in animal studies tends to occur within days of PNI, impeding translation to the timing for human chronic scenarios.
One limitation of the current study is the inclusion of various types of nerve injury, such as traumatic, diabetic, and chemotherapy-induced neuropathy.Each of these conditions can trigger distinct TR responses, as different stimuli might induce unique degeneration processes, leading to varied transcriptional profiles at different stages.Therefore, despite all presenting with a NP phenotype, the timing of transcriptional changes post-injury is likely to differ significantly among these conditions (reviewed in Cuevas-Diaz Duran et al, 2023 144 ).
The specific effects of these differing degeneration processes and their timing on the development of NP need further discussion.A thorough understanding of how different types of nerve injuries and their temporal progression influence TR and subsequent pain phenotypes is essential for elucidating the molecular mechanisms underlying NP.
The molecular techniques used could also be considered as a potential limitation.The studies that we found primarily used either RNA-sequencing Nuclease) and derivates [149][150][151][152][153] which promise to yield better results, with higher-resolution and requiring less among of sample.

CONCLUSION AND FUTURE DIRECTIONS
We found 65 research articles and selected specific ones analysing gene expression changes in various peripheral neuropathic pain models.These were used for a GO enrichment analysis to identify the shared functional profile in the PSNs.Altered gene and protein expression in PSNs post-PNI is an adaptive response aimed at functional recovery 10,11 .The genes with altered expression following PNI have functions relating to neuron projection development, signalling pathways, ion transport, and sensory perception of pain 10,30,68,72,73,76,77 .Changes in gene expression that increase neuronal excitability and spinal nociceptive input underlie development of PNP 12,154 .Moreover, failure to reverse PNIinduced TR following recovery from initial insult may result in chronic PNP 154,155 .There are numerous contentious areas within current literature regarding the expression and functional role of specific genes in PNI-induced PNP, with a need for further research elucidating the mechanisms driving these changes 10,30,68,72,73,76,77 .
Despite the limitations (stated above), the use of animal models combined with transcriptomic analysis technologies to detect PNI-induced TR may aid identification of novel therapeutic targets for PNP 2 .Elucidation of genes and regulatory mechanisms implicated in PNP presents a promising avenue for development of novel analgesics, warranting further exploration.
Finally, we propose the following directions (Table 6) for future research with the potential to help develop more effective treatments with fewer adverse effects than current therapies for PNP, a debilitating condition which remains the topic of much literature debate.background with input genes mapped to biological values for each enrichment term were calculated using the hypergeometric distribution.Significantly enriched terms (p < 0.01) were categorised into clusters based on similarities calculated using Kappa statistics to measure the strength of gene-gene interactions.Enriched terms with Kappa score > 0.3 were clustered.The most statistically significant term within each cluster is used for graphical representation, (additional terms from each cluster are listed in supplementary file 1).Terms in the heatmap are hierarchically ordered and coloured by the reversed magnitude of the hypergeometric p-values (log base 10) to represent the significance of their enrichment.
Table 4. Sensory phenotypes and transcriptomic profiles of primary sensory neuronal subtypes under physiological conditions.

Heat Shock Protein Hspb1
Growth Promoting Enzyme Odc1

Neurotransmitter inhibition Gal
Regeneration Associated Genes Gap 43

Transcription factor activator Adcyap1
Input genes were those significantly upregulated/downregulated following peripheral nerve injury 76 .Unabbreviated gene names can be found within the abbreviation list.

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
☐ The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Figure 3 .
Figure 3.A network of enriched terms designed to visualize the relationships among these terms.Terms generated from input genes significantly upregulated (A) or downregulated (B) following peripheral nerve injury.Each enrichment term is represented by a circular colour-coded node.Nodes of the same colour are part of the same hierarchical cluster.The most statistically significant term is given in the key (additional terms within each cluster can be found in supplementary file 1).The size of the node proportionally represents the number of input genes categorised to the enrichment term.The thickness of the purple edge between nodes represents the degree of similarity in genes between enrichment terms, calculated based on Kappa-statistical similarities where Kappa score < 0.3 represents significant similarity between clusters.Network visualisation analysis was undertaken with Cytoscape.

Figure 4 .
Figure 4. Heatmap summary of 'Disease Association Analysis' for differentially expressed genes following peripheral nerve injury.using the DisGeNET13 database.Input genes were those significantly upregulated (A) or downregulated (B) following peripheral nerve injury.Disease associations are hierarchically ordered and coloured by the reversed magnitude of the calculated p-values (log base 10).Analysis was done using the DisGeNET13 database.
Short operating time; continuity of nerve trunk maintained to provide optimal pathway for regeneration Number of damaged nerve fibres varies depending on degree of compression Spinal Nerve Transection Useful for stimulating phantom limb pain Lacks a local inflammatory component seen in other models; Pain behaviour J o u r n a l P r e -p r o o f Allows investigation of injured common peroneal and tibial nerves and uninjured adjacent sural nerve; easy surgical procedure; low variability in degree of damage Difficulties in performing behavioural tests; sensation in lateral paw (hypersensitive area) is difficult to test Causes robust and early neuropathic phenotype: increased thermal latency as well as decreased nerve conduction velocity and intra-epidermal nerve fibre density Associated with severe toxicity and high levels of mortality post-injection reviews of studies investigating specific neuronal clusters and their differentially expressed genes.J o u r n a l P r e -p r o o f Primary studies investigating transcription regulators and knockout studies in mice to identify changes in output gene expression.Systematic reviews on results from previous studies identifying key transcription regulators and the differentially expressed genes associated with them.Identifying precise gene function Complete understanding of genes regulating both neuronal regeneration and neuropathic pain post-PNI will further direct therapeutic options.Therapeutic interventions must consider the consequences of concomitantly affecting the protective function of reprogramming and its role in neuronal regeneration and recovery.Primary knockout studies in mice investigating the function of key genes in both regeneration and neuropathic pain.Systematic reviews of previous studies to identify those genes most specifically linked to neuropathic pain.Animal trials investigating the effect of therapeutic interventions targeting transcription regulators on both regeneration and neuropathic pain.Temporal patterns in differentially expressed genes Current studies do not sufficiently explore how gene expression changes across a variety of timepoints.Appreciation of the temporal aspects in gene expression changes will help determine optimal timing for interventions.Primary studies using scRNA-seq at multiple time points following nerve injury to assess changes in gene expression over time.Systematic reviews of previous studies utilising different time points to compare which differentially genes are more common at specific times.Hypersensitivity in primary sensory neurons Targeting genes involved in the development of hypersensitivity in primary sensory neurons may be a promising approach for the development of novel therapeutic interventions.Primary studies investigating genes involved in the development of hypersensitivity, for example those that alter expression of ion channels, receptors, or neurotransmitters to increase neuronal excitability and spinal nociceptive input.J o u r n a l P r e -p r o o f Journal Pre-proof Animal trials investigating the efficacy of novel interventions targeted as these specific genes.J o u r n a l P r e -p r o o f Journal Pre-proof

Table 1 .
Literature search terms and relevant keywords guiding study selection for this review.

Table 2 .
The methodology, strengths, and limitations of animal models of neuropathic pain.

Table 3 .
Summary of behavioural assessment methods used in animal models of peripheral neuropathy.

Table 5 .
Genes upregulated (A) and downregulated (B) in primary sensory neurones following peripheral nerve injury, cross-matched with databases of DisGeNET terms:

Table 6 .
Potential targets and future directions for the study of transcriptional reprogramming in neuropathic pain