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Abstract

Preclinical studies addressing the peripheral effects of cancer perineural invasion report severe neuronal availability and excitability changes. Oral cell squamous cell carcinoma perineural invasion (MOC2-PNI) shows similar effects, modulating the afferent’s sensibility (tactile desensitization with concurrent nociceptive sensitization) and demyelination without inducing spontaneous activity (see Part 1.). The current study addresses the electrical status (normal or abnormal) of both active (low threshold mechano receptors (LT) and high threshold mechano receptors (HT)) and inactive (F-type and S-type) afferents. Concurrently, we have also evaluated changes in the genetic landscape that may help to understand the physiological dynamics behind MOC2-PNI-induced functional disruption of the peripheral sensory system. We have observed that the altered cell distribution and mechanical sensibility of the animal’s somatosensory system cannot be explained by cellular electrical dysfunction or MOC2-PNI-induced apoptosis. Although PNI does modify the expression of several genes related to cellular hypersensitivity, these changes are insufficient to explain the MOC2-PNI-induced aberrant neuronal excitability state. Our results indicate that genetic markers provide limited information about the functional hyperexcitable state of the peripheral system. Importantly, our results also highlight the emerging role of plasma membrane Ca²⁺-ATPase activity (PMCA) in explaining several aspects of the observed gender-specific neuronal plasticity and the reported cellular distribution switch generated by MOC2-PNI.

Keywords

Cancer pain MOC2 PNI somatosensory system nociception gene expression

Introduction

TNFα and TNFR1 have also been extensively studied for their role in neuropathic pain; TNFα appears to be one of the most important cytokines released following peripheral nerve injury, leading to Schwann cell activation and neuronal sensitization.¹ This description only partially concurs with our observations, as reported in Part 1 of the current study, showing the development of a profound MOC2-induced sensitization state without any protective function on the animals lacking TNFR1 when PNI is rather advanced. However, in a broader sense, MOC2-induced injury will likely produce far deeper than reported effects on sensory neurons’ excitability and gene expression.² Two main points will be addressed during Part 2 of this study: (a) does the reported electrical status (active: HT, LT, MS and inactive: F-type and S-type) correlate with electrical changes that may question the metabolic state of the recorded afferents (and therefore its functionality) and (b) does the expression of different genes related to the cellular response to injury (hypersensitivity, cellular stress response, inflammation, calcium metabolism, etc.) correlates with the overall sensibility status of the animals (behavioral and ePhys) used in Part 1 of this study.

For example, chronic exposure to TNFα seems to alter sensory neuron excitability via TNFR1/R2 ratio change.³ Moreover, TNFR1 upregulation of several voltage-gated sodium channels (VGSC; e.g. Nav 1.8 and Nav 1.7),⁴ enhancing and accelerating channel activation.⁵ Likewise, it has been described that nerve injury may also alter voltage-gated calcium channels (VGCC; e.g. T-type Cav 3.2), increasing its density⁶ and likely forcing the enhancement of plasma membrane Ca²⁺-ATPase activity (PMCA 1-4)⁷ and modulating Ca²⁺ cellular metabolism (CCM). The current part of this study aims to clarify whether the aforementioned changes are sufficient to modulate the electrical signature of the affected sensory neurons, compromising their functions.

Moreover, TNFR1 belongs to a family of receptors that, depending on the context, can induce apoptosis (Tumor necrosis factor receptor 1-associated death domain protein or TRADD).⁸ Furthermore, during a sciatic nerve injury concurrent with pain, an increase in the expression of caspase-3 has also been observed. As we know,⁹ caspase-3 degrades DNA and cytoskeletal proteins, leading to cellular apoptosis. As a counterbalance to this process, nerve injuries also trigger concurrent cellular rescue mechanisms, such as the increased expression of activating transcription factor 3 (ATF-3), which putatively enables axonal regeneration.^10,11 Again, it seems logical to question if these fundamentally opposed cellular processes are enough to explain the overall sensibility status reported by Part 1.

Although simplistic, these examples illustrate the complexity of the sensory neuron’s cellular response to injury and the need to establish if there is a correlation between the functional (behavioral and electrophysiological) readouts and the electrical/molecular status of the recorded afferents. As indicated in Part 1, the primary goal of the current study is to expand previous observations on the effects of cancer PNI on primary afferent plasticity and the putative role of TNFRs in preventing these effects and tumor growth. To continue with this assessment will be the topic of Part 2 of the current study and includes an evaluation of potential changes in the cellular electrical properties of the recorded afferents (active and inactive) and the genes putatively related to the development (or lack thereof) of chronic pain states.

Methods

Thirty-two mice, 4–6 weeks of age, of two breeds (C57BL/6J termed wild type (WT) and Tnfrsf1a^tm1Mak (TNFR1 -/- termed KO); Jackson Laboratory, Bar Harbord, ME. USA) with four experimental groups (Sham, MOC2-PNI, males, and females, eight animals per group) were used to study the putative protective role of the absence of TNFR1 on the development of the MOC2-PNI-induced neuropathy. Cell culture procedures and MOC2 cell implantation on the animals’ sciatic nerves were performed at New York University (NYU) facilities. They were then transported to Wake Forest University (WFU) for behavioral, electrophysiological (sensorial and biophysical), and molecular evaluation following a linear consecutive experimental design (Figure 1(a)). Due to its size and detail our study will be presented in two parts: (1) Animals behavior (reflexive and non-reflexive) and peripheral sensory electrophysiology (distribution, sensibility, and afferents conduction velocity (CV); Part 1; Figure 1(a) gray box) and (2) peripheral sensory neurons somatic biophysics (active and passive) and gene expression (Part. 2). In both facilities, the animals were housed in pairs under a 12-h light/dark cycle in a climate-controlled room. The use and handling of animals were under guidelines provided by the National Institutes of Health and the International Association for the Study of Pain, and the Institutional Animal Care and Use Committees of New York University and Wake Forest University Health Sciences approved the procedures and experiments.

Figure. 1.

(a) Schematic of the experimental design performed by New York University (NYU) and Wake Forest University (WFU) (C: contralateral, I: ipsilateral), (b) Representative photo of the SN with and without MOC2 tumor (I.) and overall IHC of the area of contact between the SN and the MOC2 tumor (II.).

Cellular somatic properties

Somatic active electrical properties (SAEP)

The active membrane properties of all excitable neurons were analyzed in APs and obtained during RF characterization. These parameters included per AP: Amplitude (mV), duration at 50% of the amplitude (D50, ms) of the AP, after-hyperpolarization amplitude (AHP, mV), and AHP duration at 50% of the AHP amplitude (AHP50, ms), along with the maximum spike depolarization rate (MDR, dV/s) and repolarization rate (MRR, dV/s). AP and AHP durations were measured at half amplitude (D50 and AHP50, respectively) to minimize hyperpolarization-related artifacts.

Somatic passive electrical properties (SPEP)

Passive membrane properties were analyzed in all neurons by injecting either negative current (9 pulses; 500 ms pulse, 0–1.9 nA step 0.1) to measure input resistance (Ir, MΩ), time constant (τ, ms), and positive current to measure the membrane rheobase (mv), throughout balanced electrodes.

Immunohistochemistry (IHC)

To visualize the perineural invasion of MOC2, tumors were collected 3 weeks after implantation, and the tissue was analyzed using IHC (n = 3; Figure 1(b) I. and II.) following electrophysiology. Mice under anesthesia (see 2.3) were transcardially perfused with 0.1 M phosphate buffer (PB, ph 7.4) followed by 4% paraformaldehyde. Tumors were post-fixed overnight submerged in 4% paraformaldehyde in PB, then transferred after 24 h into PB and stored at 4°C. Afterward, the tissue was washed in PB and immersed in 30% sucrose at 4°C for cryoprotection until sectioned on a cryostat. Tumor sections (50 μm) were collected on slides and stored at −80°C until processed. Sections were processed simultaneously with antibodies for Substance P^7,12 and PGP9.5 antibodies. PGP 9.5 and SP were used to visualize the totality of the fibers from the sciatic nerve and afferents with slow conduction velocity from peptidergic nociceptive neurons, respectively, confined within the tumor’s vicinity. DAPI helped to visualize the nuclei of MOC2 and other cellular components of the tumor microenvironment. Sections were washed with 0.01 M phosphate buffer saline (PBS) with 0.1% Triton X-100 (PBST), incubated for 1 h in blocking solution (3% normal donkey serum (NDS; # 017-000-121, Jackson Immuno Research Labs, West Grove, PA, USA) in PBST and overnight at 4°C in 1.5% NDS with rat anti-SP (1:500, #556312, BD Biosciences, San Jose, CA, USA) and rabbit anti PGP9.5 (1:1000,# CL7756AP, Cedarlane, Burlington, NC, USA). Afterward, sections were washed three times for ten minutes with PBS and incubated for 2 h at room temperature with donkey anti-rat Cyanine 3 and donkey anti-rabbit Cyanine 2 (1:400, Jackson Immuno Research Labs, West Grove, PA, USA). Finally, sections were incubated for 5 min with 4′,6-diamidino-2-phenylindole (DAPI, 1:10000, D21490, Invitrogen, Molecular Probes, Eugene, OR, USA) washed thoroughly in PBS, mounted on plus-slides, air-dried, dehydrated in ethanol, cleared in xylene, and cover slipped with DPX mounting media. Sections were examined, and images of selected sections of tumors were acquired at 20–60X magnification using an Olympus FV1200 confocal microscope with Olympus Fluoview Version 4.2b software. (Olympus Corporation of the Americas, PA, U.S.A).

mRNA measurement

After the ePhys evaluation, animal tissues were collected for mRNA measurements. MOC2 tumors, a similar lengthy portion of the sciatic nerve (ipsilateral and contralateral), and the ganglia (L4 and L5, ipsilateral and contralateral) were dissected. The mRNA measurements were aimed at specific markers. First, we evaluated the effects of the tumor on the expression of putative ganglionic cellular markers for inflammation (tumor necrosis factor receptors (Tnfr1, Tnfr2)), hypersensitivity (protease-activated receptor 2 (Par2), tachykinin precursor 1 (Tac1)), calcitonin/calcitonin-related polypeptide alpha (Calca), plasma membrane Ca²⁺ ATPases 1-4 (Pmcas 1-4), cellular stress response (activation transcription factor 3 (Atf3)), and apoptosis (caspase-3 protein (Casp3); Table 1). Secondly, we evaluated the effects of the tumor on the sciatic nerve markers for inflammation (tumor necrosis factor-alpha (TNFα), purinergic receptor 3 (P2X3), interleukin-6 (IL-6)), myelination (myelin protein zero (Mpz) and myelin binding protein (Mbp)), tachykinins (TK), and hypersensitivity (tachykinin precursor 1 receptor (Tac1R); Table. 2). Glyceraldehyde 3-phosphate dehydrogenase (GAD-PH) was used as a control in all cases.

Table 1.

L4-L5 ganglia gene primers and cellular markers.

Function	Gene	Protein	Cite function	Primer sequence 5’-3’
Inflammation (IF)	Tnfrsf1a	TNFR1	^13,14	GCA AAG ACC TAG CAA GAT AAC C GTG AGT GCG TCC CTT GC
Inflammation (IF)	Tnfrsf1b	TNFR2	^4,14,15	GCC TTC CTG TCA TAG TAT TCC TG TCT TCG AAC TGC AGC TGT G
Hypersensitivity (HS)	F2rl1	PAR-2	¹⁶	TGG CCA TTG GAG TCT TCC TGT T TAG CCC TCT GCC TTT TCT TCT C
	Tac1	TK	¹⁷	TCT TTC GTA GTT CTG CAT CGC AGG CTC TTT ATG GAC ATG GC
	Calca	CGRP	¹⁸	AAC CTT AGA AAG CAG CCC AGG CATG GTG GGC ACA AAG TTG TCC TTC ACC A
	P2rx3	P2X3	¹⁹	TCT CCA GCA GAG ACA TCA GCA GGG AGC ATC TTG GTG AAC TCA G
Cellular stress response (S)	Atf3	ATF-3	²⁰	AGA TGT CAG TCA CCA AGT CTG TGT CTT CTC CTT TTT CTT GTT TCG
Cellular stress response (S)	Casp3	CASP-3	²¹	GGA CTG GAT GAA CCA CGA C GAC TGA TGA GGA GAT GGC TTG
Ca²⁺ cellular metabolism (CCM)	Atp2b1	PMCA-1	^7,22,23	CCG TTT CTC CTT CAC CTT CT AAT TGC AGC CAT AGT ATC ATT GG
	Atp2b2	PMCA-2	^7,22,23	CTT CTG CTT CAC CTT CAT CCT C ACT GTC CTT CTA CCA CCC A
	Atp2b3	PMCA-3	^7,22,23	GTT GTC TCC AGT CAC CAT ACG GAC CTT ACC TGC ATA GCT GTT
	Atp2b4	PMCA-4	^7,22,23	TCT CCT TGT CCT CAC TGT CG CCC CTG AAA ATC GCA ACA AAG
Control	Gapdh	GAPDH	²⁴	GGC TCA TGA CCA CAG TCC CAC ATT GGG GGT AGG AAC AC

Table 2.

Sciatic nerve gene primers and cellular markers.

Function	Gene	Protein	Cite function	Primer sequence 5’-3’
Inflammation (IF)	Tnf	TNF-α	⁴	TCT TTG AGA TCC ATG CCG TTG AGA CCC TCA CAC TCA GAT CA
Inflammation (IF)	Il6	IL-6	²⁵	TCC TTA GCC ACT CCT TCT GT AGC CAG AGT CCT TCA GAG A
Myelination (MY)	Mbp	MBP	¹⁹	CCT CCG TAG CCA AAT CCT G ACC CAA GAT GAA AAC CCA GTA G
Myelination (MY)	Mpz	MPZ	¹⁹	ATA GCT GGG GTA GGG CAA GTT TCA TGT TGG TAG CAG TGG
Hypersensitivity (HS)	Tacr1	NK1R	²⁶	AGT AGA TCA GCA CAG TCA CAC GCT ACT ACT CCA CCA CAG AGA
Control	Gapdh	GAPDH	²⁴	GGC TCA TGA CCA CAG TCC CAC ATT GGG GGT AGG AAC AC

Tissue collection and RNA extraction

Tissue was collected after electrophysiological experiments from all mice except three mice used for IHC (see 2.4). To collect tissue, mice deeply anesthetized were transcardially perfused with 0.1 M phosphate buffer (PB, ph. 7.4). L4 and L5 DRGs and sciatic nerves (ipsilateral and contralateral to MOC2) were dissected and rapidly flash-frozen on dry ice to preserve RNA integrity. The total RNA was extracted from the tissue using the ReliaPrep RNA tissue Miniprep System (# Z6111, Promega, Madison, WI, USA) following the manufacturer’s protocol. Contaminating genomic DNA was removed by DNase I digestion. The quality and concentration of RNA samples were assessed using a NanoDrop 2000c spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA).

Reverse transcription

Reverse transcription was performed using a High-capacity cDNA reverse transcription kit (# 4368814, Applied Biosystems, Foster City, CA, USA). Total RNA (100 ng) was converted to single-stranded cDNA. Reverse transcription without reverse transcriptase was also performed to assess genomic DNA contamination.

Quantitative real-time polymerase chain reaction (qPCR)

Primers (listed in Tables 1 and 2) were designed to minimize amplification from contaminating genomic DNA (Integrated DNA Technology Inc., Coralville, Iowa, USA). qPCR was performed using LightCycler 480 SYBR Green I Master (# 04707516001, Roche Diagnostics GmbH, Mannheim, Germany) in a 384-well PCR Microplate (#PCR-384-LC480WNFBC, Axigen, UT, USA). PCR reactions contained 500 nM of primers and 20 ng of reverse-transcribed total RNA in 10 μL. qPCR was performed with an initial 5 min pre-incubation at 95°C followed by 45 cycles of amplification (10 s at 95°C, 10 s at 60°C, and 10 s at 72°C). Melting curve analysis was performed at the end of qPCR and showed a single peak for each gene in each sample. Relative quantification analysis (the ratio of a target DNA sequence to a reference DNA sequence (GAPDH) was performed using the second derivative maximum method as previously described.²⁷ Samples containing no cDNA template and no reverse transcriptase were run as negative controls for contamination and amplification of genomic DNA, respectively. All samples were run in duplicates. The mRNA levels of every gene were normalized to mRNA levels of GAPDH in each sample.

Statistical analysis

Before analysis, parametric assumptions were evaluated for all variables using histograms, identifying outliers with boxplots, descriptive statistics, and the Shapiro–Wilk test for normality. Data are reported as median (range or quartiles) if not normally distributed or mean (standard error) if normally distributed. Student’s t-test and repeated measures analysis of variance (ANOVA) were used for normally distributed data, and the Friedman test and Mann Whitney U-test were used for not normally distributed data. Changes in Em in A-HT over time were analyzed using repeated-measures ANOVA with Greenhouse & Geisser sphericity correction as distributions at each time point proved parametric, and there were no significant outliers. Friedman tests were run on the number of APs per stimuli and duration data as the distributions were non-parametric at one or more time points in each dependent variable. Gene expression analysis was conducted with GraphPad Prism 9.4 software (Brown-Forsythe test, Bartlett test, Analysis of Variance (ANOVA), and Bonferroni’s multiple comparison test). For all analyses, p was set at 0.05 for statistical significance. All post hoc analyses were Bonferroni adjusted. Analyses were conducted using SPSS Statistics for Windows, version 22 (IBM Corp, Armonk, NY), and OriginPro 9.5 (Northampton, MA).

Results

Effect of MOC2-PNI on the afferent’s somatic electrical properties

Active electrical properties

The spikes generated by the cellular body of sensory neurons with mechanical RF in different modalities (tactile (LT) and nociceptive (HT)) were analyzed and compared with cells without RF (F-type and S-type) correlating four different parameters: AP duration (D50, ms) versus AP amplitude (mV) and AHP duration (AHP50, ms) versus AHP amplitude (mV). As presented in Figure 2(a), the spikes of both modalities in male WT animals show highly stereotyped correlations. AP duration (D50, ms) versus AP amplitude (mV): While LT was characterized by narrow (D50: 0.7 ± 0.1 ms), low amplitude (46.6 ± 6.3 mV) spikes, HT presented significantly (p < 0.001) broader (D50: 2.1 ± 0.3 ms) with significantly (p < 0.01) larger amplitude (61.7 ± 12 mV). Although some outliner cells were observed for one independent parameter (D50 or amplitude), no outliner was observed for its combination. Based on this data, 100% of the analyzed cells classified as LTs were located below 1.2 ms (D50) and 55 mV (amplitude), while 100% of the analyzed cells classified as HT were above 1.2 ms (D50) with some dispersion in the amplitude (85%) but largely above 55 mV. AHP duration (AHP50, ms) versus AHP amplitude (mV): Although less robust than the spike shape, the AHP was also significantly different between modalities. LT showed significantly (p < .05) shorter AHP durations (AHP50: 1.7 ± 0.5 ms) and smaller amplitude (8 ± 1 mV) than HTs (AHP50: 8.3 ± 0.9 ms, amplitude 13.5 ± 1.5 mV). Both mechanically sensitive and insensitive cell data indicate that LT has 100% consistency with an F-type action potential. In comparison, HT is 100% consistent with an S-type, except for shorter AHP amplitude, significantly (p < 0.05) larger in S-type cells. As presented in Figure 2(b) to (d), this overall description is consistent across genders and strains, with a sole punctual difference in the female KO group. No statistical difference exists between the AHP (duration and amplitude) from HT cells and S-types in these animals.

Figure 2.

MOC2-PNI-induced silencing does not affect the correlation between spike shape (SAEP) and modality. Correlation of the spike shape (AP and AHP) and function in both mechano-sensitive (tactile: LT (black circle); nociceptive: HT (both CVs; purple triangle)) and mechano-insensitive afferents (F-type (black open box) and S-Type (purple crossed box)) per gender (a and c) and strain (b and d), in sham and MOC2-PNI (green), animals.

Finally, as observed in Figure 2(a) to (d), MOC2-PNI didn’t modify the spike shape (D50 and/or amplitude) or significantly change the correlation between cellular modality and AP type. However, these animals (MOC2-PNI) presented the shorter HTs/S-type spikes of the group (female WT) and the shorter AHP duration (male KO).

Passive electrical properties

In male WT sham animals tactile afferents (LT) showed significantly (p < 0.01) lower membrane potential (Em: −59.7 ± 2.6 mV) and input resistance (Ri: 103 ± 32 MΩ) than nociceptive afferents (HT; Em: −49 ± 1.9 mV; Ri: 193 ± 32.2 mΩ), while in cells without RF (F- and S-type) both parameters were very similar. MOC2-PNI induces a significant (p < 0.05) reduction in the Em (to −57 ± 3.4 mV) and Ri (to 96 ± 10 MΩ) of HT afferents, rendering these parameters indistinguishable between cellular subtypes (with or without RF; Figure 3(a)). Oppositely, male KO sham animals showed no differences between cellular groups. However, the mechanosensitive recorded cells displayed a similar correlation between modality and these parameters, with LT showing a significantly (p < 0.01) lower Em (−60.4 ± 1.7 mV) and Ri (75 ± 18 MΩ) and a significant (p < 0.05) reduction on the Ri of F-type cells (Figure 3(b)). Female animals presented similar tendencies but with larger variability and only the Ri on female WT animals was significantly (p < 0.01) different between tactile (LT) and nociceptive (HT) afferents in both strains and treatment (Figure 3(c) and (d)). No significant differences between groups were observed in Tau (ms) or the cellular rheobase (data not shown).

Figure 3.

Restricted passive properties (SPEP: Em and Ri) are susceptible to MOC2-PNI disruption and can be used to define modality in a gender-specific, strain-specific manner. Effects of MOC2-PNI on the SPEP of mechano-sensitive (LT and HT) and mechano-insensitive (F-type and S-type) of males (a and b) and females (c and d) of both strains (WT and KO).

Effects of MOC2-PNI on mRNA expression

The results of this analysis have been grouped to provide a comprehensive description of the effects of MOC2-PNI on the ganglia and the nerves of both genders and strains. Aiming to define if gender and strain induce a differential gene expression, we performed a stratified data analysis. First, the overall gene expression was evaluated using normalized data (heatmap; Ipsilateral data normalized to contralateral). Following this general evaluation, a secondary analysis of the absolute values (non-normalized) was performed if a significant effect was detected using normalized data.

Effects of MOC2-PNI on gene expression of normalized data (heatmap)

At the ganglia:

Inflammation (IF)

Tnfrsf1a was not expressed (Cp > 35) in KO mice when compared to WT (Cp < 30). MOC2-PNI significantly (p < 0.05) increased the expression of ganglionic Tnfrsf1a (Figure 4(a)) within WT females only; by contrast, it induced a significant (p < 0.001) increment in the mRNA expression of Tnfrsf1b in male KO animals only.

Figure 4.

MOC2-PNI modulates the mRNA expression in both the L4 ganglia and the sciatic nerve toward hypersensitivity and hyperexcitability in a gender-specific and strain-specific manner. Effects of MOC2-PNI on the mRNA expression of several genes related to inflammation (IF), hypersensitivity (HS), cellular stress response (S), and Ca²⁺ cellular metabolism (CCM), myelination (MY) in both genders and strains. (a) In the L4 ganglia and (b) in the nerve.

Hypersensitivity (HS)

The presence of MOC2-PNI affected the mRNA expression values of only one gene ( F2rl1 ) within males WT, while the rest remained unchanged ( Tac1, Calca, and P2rx3 ). MOC2-PNI induces a significant (p < 0.05) increase in the mRNA expression of F2rl1 in a gender and strain-specific manner (male WT only; Figure 4(a)).

Cellular stress response (S)

The mRNA expression of Atf3 was significantly (p < 0.01) different within females WT only (Figure 4(a)). However, when absolute values of the data were compared, several significant differences were observed (see 3.2.2.) in the basal level of expression of this gene, its response to MOC2-PNI, and between genders (see below non-normalized analysis, Figure 5(d)).

Figure 5.

MOC2-PNI modulates the mRNA expression of the L4 ganglia toward inflammation, hypersensitivity, and cellular stress, affecting both contralateral and ipsilateral ganglia in a gender-specific and strain-specific manner. Contralateral (C) and ipsilateral (I) MOC2-PNI-induced modulation of mRNA expression in susceptible injury-related genes (see Figure 6; a–d per gene) in males and females of both strains (WT and OK) and conditions (sham vs PNI), (e) Casp3.

As observed in Figure 4(a), both genders show a MOC2-PNI-induced significant (p < 0.05) increase in this Casp3 expression. Notably, sham surgery exerted a significant (p < 0.05) effect on female animals (see below non-normalized analysis, Figure 5(e)).

Ca²⁺ cellular metabolism (CCM)

MOC2-PNI also modulated the RNA expression of the Atp2b gene. This modulation was largely gender-specific, affecting only males. Male KO sham animals have significantly higher mRNA expression levels of this gene, compared to WT (except Atp2b2 is significantly higher (p < 0.05) in male WT sham). In addition, the presence of MOC2-PNI substantially reduces the expression of Atp2b1 , Atp2b3 , and Atp2b4 in KO animals (Figure 4(a)).

At the injury site:

Inflammation (IF)

MOC2-PNI induces gender and strain-specific effects in the expression of Tnf . This gene significantly (p < 0.01) increases in males KO and females WT PNI (Figure 4(b)). On the other hand, MOC2-PNI induces a significant (p < 0.01) increase of Il-6 expression in all groups but male KO animals (Figure 4(b)).

Myelination (MY)

Although a clear axonal regeneration process was observed in all groups (see 3.2.1.3.), neither gender, strain, nor the presence of MOC2-PNI induced a differential expression of Mbp or Mpz’s mRNA at the injury site (Figure 4(b)).

Hypersensitivity (HS)

MOC2-PNI did not induce significant changes in the expression of Tacr1 in any of the experimental groups (Figure 4(b)). Conversely, robust sex-specific differences were observed when comparing absolute values (Figure 7 see supplementary data).

Effects of MOC2-PNI on gene expression absolute values of gene expression by comparing non-normalized data

Inflammation (IF)

Consistent with the normalized data (heatmap 3.2.1.1), MOC2-PNI induces a significant (p < 0.05) increase in Tnfrsf1a expression within females (Figure 5(a)). However, in addition to the observed effects of MOC2-PNI on the expression of Tnfrsf1b within males KO (3.3.1.1), a significant (p < 0.05) increase in the expression of this gene was also observed in the contralateral side females KO (Figure 5(b)).

Hypersensitivity (HS)

Consistent with the normalized data (heatmap 3.2.1.2), MOC2-PNI induces a significant (p < 0.01) increase in the expression of F2rl1 , but only on males KO (Figure 5(c)).

Cellular stress response (S)

In addition to the data obtained by normalization (heatmap 3.2.1.3), we also observed (non-normalized data) that the basal expression levels of Atf3 were significantly (p < 0.01) different between genders (extremely low in males by comparison with females WT). While sham surgery significantly (p < 0.01) increases this gene expression in females WT, MOC2-PNI modulated the expression of this gene in a gender-specific, strain-specific manner. The expression of Atf3 significantly (p < 0.05) increased in male WT; meanwhile, it was significantly reduced in males KO (p < 0.05) and females WT (p < 0.001; Figure 5(d)). Consistent with the normalized data (heatmap 3.2.1.3), MOC2-PNI induced a significant increase in the expression of Casp3 in male WT (p < 0.05), male KO (p < 0.01) and females WT (p < 0.05) but not females KO (Figure 5(e)).

Ca²⁺ cellular metabolism (CCM)

As presented in Figure 6, the mRNA expression of these genes varies between genders, strains, and their response to MOC2-PNI. Atp2b1 basal expression was significantly (p < 0.001) lower in males WT (compared to males KO, females WT, and females KO). However, MOC2-PNI induced a significant (p < 0.01) decrement in this gene expression only in females WT (Figure 6(a)). On the other hand, Atp2b2 showed similar basal mRNA expression across genders and strains, with a significant (p < 0.05) reduction of its expression only in males WT PNI (Figure 6(b)). Similar to Atp2b1, males WT showed significantly (p < 0.01) lower basal expression of Atp2b3 and Atp2b4 (compared to males KO, females WT and females KO). However, a gender-specific, strain-specific response to MOC2-PNI was observed. While males WT showed significant (p < 0.05) increases in the expression of these genes, females showed a significant (p < 0.001 and p < 0.05, respectively) reduction, with no effect on males or females KO. (Figure 6(c) and (D)).

Figure 6.

MOC2-PNI modulates the mRNA expression of the L4 ganglia toward hyperexcitability and Ca2⁺ metabolic stress, affecting both contralateral and ipsilateral ganglia in WT animals (both genders) but not KO. Contralateral (C) and ipsilateral (I) MOC2-PNI-induced modulation of mRNA expression in susceptible hyperexcitability-related genes (see Figure 6; a–d per gene) in WT and KO animals of both genders, strains (WT and OK) and conditions (sham vs PNI).

Discussion

Studies addressing rodents’ transition from acute to chronic pain have consistently reported the same overall results. Rapidly induced tactile desensitization concurrent to nociceptive sensitization (early response) and a decline in afferents available for activation (late response) almost unavoidably occur in a two-staged manner. The chosen model only modifies the magnitude of these modality-specific changes in sensibility and the overall afferent trajectories toward deactivation.^17,28–35

Like these reports, MOC2-PNI injury also produces a dramatic switch in the availability and sensibility of active afferents (LT and HT; Part 1). However, neither of these studies has evaluated the electrical status or the changes in the expression of injury-related genes after this process has concluded and the full neuropathy has developed. The current study addresses this caveat, indicating that the deactivated afferents are not damaged and all electrical parameters remain within a normality status. Furthermore, the current study revealed that gender and strain (WT vs KO) exerted differential effects on the expression of genes widely used as indicators of pathological processes (MOC2-PNI). These changes will be discussed independently per gene and gender.

What is behind the PNI-induced mechanic-sensitive afferents distribution switch?

The simplest explanation for the increased number of mechano-insensitive afferents presented in Part 1 of this study lies within a combination of potential nerve blocking and cell damage as a direct consequence of MOC2 tumor growth. Nerve compression could potentially induce motor and sensory dysfunctions (e.g. numbness and pain)³⁶ and terminally damage the cells (inflammation, autophagia, and apoptosis).¹²

However, in this study, the overall analysis of our recordings offers no evidence of such degeneration or A fibers block. Although some evidence of PNI-induced cellular inflammation was observed (reduction of the Ri and lower Em), these effects were only restricted to the WT group. Nevertheless, the recorded mechanically insensitive afferents (F-type and S-type) were mostly electrical sound (spike shape), clustering close to their putative modalities (tactile and nociceptive)^37-40 with almost 100% of parity above around two evoked spike thresholds (−55 mV of AP amplitude and 1.2 ms of AP duration). Importantly, we also detected unusually long AHP durations for insensitive afferents, a parameter usually related to hyperactivation³⁵ due to its importance in regulating the cellular refractory period and discharge frequency.⁴¹

These data strongly support that the mechanically unresponsive afferents are not damaged, which raises the question of whether the distribution switch is due to a physiological deactivation process to compensate for large changes in nociceptive excitability that may compromise animals’ survival. Although nerve block cannot be completely discarded in this particular model (PNI), our group’s historical observations indicate the validity of the deactivation process.^17,28–35

Do the changes in the genetic expression recapitulate the gender-specific and strain-specific behavioral and ePhys effects induced by MOC2-PNI?

Changes in the expression of DRG genes related to neuropathic pain have been extensively studied in rodent models.⁴² Regardless of the robustness and reproducibility of rodents’ chronic pain behaviors,⁴³ tissue heterogeneity and bi-compartmentally (peripheral and central) increases the complexity of interpreting neuropathic pain gene signatures at the DRGs.

The current study did observe limited consistency when attempting to correlate the expression of some of the analyzed genes and their alleged function, as follows. TNF: it has been argued that this cytokine (produced by the tumors) and its receptor (TNFR1) have a pivotal role in peripherally mediated hyperalgesia,⁴ demyelination,⁴⁴ and apoptosis.²¹ These effects have been attributed to the activation of the immune system (monocytes and macrophages) toward demyelination due to Schawn cell TNFR1 activation,⁴⁵ but more importantly, due to direct modulation of Na⁺ channel’s expression in the sensory neurons, thus enhancing voltage-gated Na⁺ current (NaV 1.7–1.9) and altering their electrical responsiveness.⁴⁶

In the present study, we did observe some protective effects of the absence of TNFR1 against the mild sensitization induced by sham surgery (behavior). However, contrary to the observation of Del Rivero et al.,¹³ gender was not a factor (Part 1. Cellular distribution) in the modulation of this effect. Furthermore, when absolute values were analyzed, increased mRNA expression of TNFα and TNFR1 and decreased TNFR2 expression were observed within female mice with tumors, which may explain why more successful attempts of blocking TNFα-induced neuropathic pain are being achieved in males but not in females.¹³ However, in the presence of advanced MOC2-PNI, the absence of TNFR1 seems to increase the hyperalgesic effects of MOC2 and general cellular deactivation. Consistent with these data, no major gender or strain-specific changes were observed in the expression of hypersensitivity/demyelination markers (Tac1 (SP), Calca (CGRP), and P2rX3 (P2X3)¹⁹ (ganglia) or Mbp, Mpz, and Tacr1 (NK1R; nerve)).

As expected, females show a predominant differential mRNA expression of IL-6,⁴⁷ with increased levels in both strains (WT and TNFR1 KO). By contrast, IL-6 only increased within WT males. Our data concur with previous observations indicating that TNF administration dramatically increased IL-6 production (after 6 h) but not in TNFR1 KO mice.⁴⁸

Several methodological differences in the model and the advanced stage of MOC2-PNI can explain this discrepancy. However, we did observe an increase in the expression Tnfrsf1b (TNFR2) in male KO mice, which may explain why males react differently to the TNRF1R inhibition.¹³ Tnfrsf1b (TNFR2) is believed to exert neuroprotective effects.^15,45 Although restricted to certain T-cell populations, endothelial cells, microglia and specific neuron subtypes, oligodendrocytes, cardiac myocytes, thymocytes, and mesenchymal stem cells, this receptor does coexpress with Tnfrsf1a (TNFR1).⁴⁹ Moreover, this coexpression has suggested that anti-TNF analgesic effects may be associated with a reduced TNFR1/TNFR2 ratio.⁵⁰ Our results support this association and, to some extent, the reported gender-specific effects of TNFR1 inhibition.¹³ However, even at this extreme ratio (absence of TNFR1), the over-expression of Tnfrsf1b (TNFR2) was insufficient to prevent the effects of MOC2-PNI within our time point.

Informed by the ePhys data (Part 1.), we expected differential expression of genes encoding for neurogenic inflammation and regeneration. F2rl1 (Par2) has been related to the release of CGRP and SP as part of the peripheral system’s neurogenic inflammatory response.¹⁶ As such, we expected increased expression of F2rl1 in the MOC2-PNI animals. Surprisingly, this gene’s expression only increased within male mice with tumors. This seems to correlate with our observation that in male WT animals with tumors, the distribution of LT afferents (but not nociceptive) is unaffected by MOC-PNI. In the same way, Atf3 (ATF3), a gene related to axonal injury and regeneration,²⁰ is also expressed in male WT sham but not in females or KO animals. Furthermore, MOC2-PNI-induced injury significantly reduces Atf3 expression in a manner that correlates with the ePhys data and effectively blocks the ATF3-mediated regenerative process, otherwise very active in sham animals (females).

Although the correlation between the low expression of F2rl1 and Atf3 seems logical (less regeneration may be needed in the presence of mild nerve injury), the uncorrelated changes between the overexpression of F2rl1, the lack of changes in the expression of Calca (CGRP) or Tac1 (SP) and the under expression of Atf3 is puzzling. This inconsistency may be explained by the fact that most previous studies based their analysis on normalized data (as represented in heat maps). However, when absolute values are analyzed, ATF3 increased ipsilaterally to tumors (males WT only), and TAC1 increased both ipsilateral and contralateral to tumors within WT males (see Figure 7 supplementary data). Moreover, the ongoing cellular apoptotic process (Casp3 (Caspase-3)) seems similar in all groups, suggesting an important disconnection between physiological function and gene expression. While transcription-dependent changes in gene expression are important, recent work demonstrates that activity-dependent regulation of mRNA translation is key to controlling the cellular proteome and developing and maintaining persistent pain.⁵¹

Is Ca²⁺ metabolism informative about cellular activity (or lack thereof)?

In a broad sense, voltage-gated Ca²⁺ channels (VGCCs) represent one of the most important regulators of Ca²⁺ concentration in neurons,⁵² and they play an important role in the peripheral system’s response to injury.⁵³ Although essential to cell function and survival,⁵⁴ intracellular Ca²⁺ overload can lead to apoptosis (by sustained Ca²⁺ influx) or necrotic lysis (by rapid Ca²⁺ influx). Thus, in neurons, Ca²⁺ intracellular concentration is under exquisite control by either Na⁺/Ca²⁺ exchangers (NCX) and ATP-driven plasma membrane Ca²⁺ pumps (PMCAs).⁵⁵ The latest (PMCAs 1-4) have been linked to neuronal hyperexcitability after nerve injury.²²

Although NCX exchangers were not explored, Atp2b’s (PMCA’s) ganglionic expression was indeed deeply modulated by MOC2-PNI. Similarly to Atf3 (ATF3), the expression of PMCAs was constitutively low in male WT (1, 3, and 4) but not in females, and its expression was driven in opposite directions. Similarly, as reported by Ogura et al.’s,⁷ injury increased the overall expression of Atp2b’s (PMCA’s) in male animals while decreasing its expression in females. To the best of our knowledge, this is the first report that revealed as well that not only gender but also the lack of TNFR1 affects the expression of PMCAs.

Although the literature does not provide evidence of a direct connection between these genes (Atf3 (ATF3), and Atp2b’s (PMCA’s)), low levels of expression seem to indicate a natural resilience in male animals’ peripheral systems to reach a hyperexcitable state. In the same way, the correlation between gender and differential expression of Atp2b’s (PMCA’s) seems to suggest a different cellular susceptibility to activation, explaining several (but not all) observations related to gender-specific responses to injury.⁵⁶ Further, physiological studies aiming to correlate the deactivation process and the concurrent changes in gene expression are required.

Conclusions

The deactivation process (or lack thereof) requires corroboration. Clearly, some of the analyzed genes are activity-dependent (e.g. Atp2b’s (PMCAs)) and may help establish the functional status of the peripheral system response (active or inactive). Due to the abrupt, uncertain nature of peripheral cell deactivation induced by MOC-PNI and several other models, it seems clear that the mRNA expression alone does not address the cellular physiological state, and high variance is to be expected in similar studies.

Supplemental Material

sj-docx-1-mpx-10.1177_17448069251323666 – Supplemental material for Advanced cancer perineural invasion induces profound peripheral neuronal plasticity, pain, and somatosensory mechanical deactivation, unmitigated by the lack of TNFR1. Part 2. Biophysics and gene expression

Supplemental material, sj-docx-1-mpx-10.1177_17448069251323666 for Advanced cancer perineural invasion induces profound peripheral neuronal plasticity, pain, and somatosensory mechanical deactivation, unmitigated by the lack of TNFR1. Part 2. Biophysics and gene expression by Silvia Gutierrez, Renee A Parker, Morgan Zhang, Maria Daniela Santi, Yi Ye and Mario Danilo Boada in Molecular Pain

Footnotes

Acknowledgements

In the loving memory of Rafaela Endara-Maldonado and Juan Bernardo Boada-Bustos.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Supported by grants R01 DE029493 to YY and 1P01NS119159-01A1 to MDB from the National Institutes of Health, Bethesda, MD, USA.

ORCID iD

Mario Danilo Boada

Supplemental material

Supplemental material for this article is available online.

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Supplementary Material

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Advanced cancer perineural invasion induces profound peripheral neuronal plasticity,pain,and somatosensory mechanical deactivation,unmitigated by the lack of TNFR1. Part 2. Biophysics and gene expression

Abstract

Keywords

Introduction

Methods

Cellular somatic properties

Somatic active electrical properties (SAEP)

Somatic passive electrical properties (SPEP)

Immunohistochemistry (IHC)

mRNA measurement

Tissue collection and RNA extraction

Reverse transcription

Quantitative real-time polymerase chain reaction (qPCR)

Statistical analysis

Results

Effect of MOC2-PNI on the afferent’s somatic electrical properties

Active electrical properties

Passive electrical properties

Effects of MOC2-PNI on mRNA expression

Effects of MOC2-PNI on gene expression of normalized data (heatmap)

Inflammation (IF)

Hypersensitivity (HS)

Cellular stress response (S)

Ca2+ cellular metabolism (CCM)

Inflammation (IF)

Myelination (MY)

Hypersensitivity (HS)

Effects of MOC2-PNI on gene expression absolute values of gene expression by comparing non-normalized data

Inflammation (IF)

Hypersensitivity (HS)

Cellular stress response (S)

Ca2+ cellular metabolism (CCM)

Discussion

What is behind the PNI-induced mechanic-sensitive afferents distribution switch?

Do the changes in the genetic expression recapitulate the gender-specific and strain-specific behavioral and ePhys effects induced by MOC2-PNI?

Is Ca2+ metabolism informative about cellular activity (or lack thereof)?

Conclusions

Supplemental Material

sj-docx-1-mpx-10.1177_17448069251323666 – Supplemental material for Advanced cancer perineural invasion induces profound peripheral neuronal plasticity, pain, and somatosensory mechanical deactivation, unmitigated by the lack of TNFR1. Part 2. Biophysics and gene expression

Footnotes

Acknowledgements

Declaration of conflicting interests

Funding

ORCID iD

Supplemental material

References

Supplementary Material

Ca²⁺ cellular metabolism (CCM)

Ca²⁺ cellular metabolism (CCM)

Is Ca²⁺ metabolism informative about cellular activity (or lack thereof)?