TSPO PET selectively detects the denervated skeletal muscle district remodeling after experimental peripheral nerve injury

Abstract

Background

Peripheral nerve injury induces inflammatory and stress-related remodeling in downstream skeletal muscle before overt atrophy develops, yet non-invasive approaches capable of detecting and anatomically localizing early muscle involvement remain limited. The 18-kDa translocator protein (TSPO), a marker of inflammatory activation and mitochondrial stress, is a molecular target for PET imaging with [¹⁸F]DPA-714. This study aimed to determine whether TSPO-targeted PET imaging enables early and spatially resolved detection of denervation-induced skeletal muscle injury distal to a peripheral nerve lesion.

Results

Adult mice underwent unilateral sciatic nerve crush. One-week post-injury, static [¹⁸F]DPA-714 PET imaging was performed to assess tracer uptake in hindlimb skeletal muscle. Uptake was quantified and compared between injured and contralateral limbs as well as with sham-operated mice. Ex vivo validation included CD68 immunofluorescence, Western blot analysis of TSPO expression, and laminin-based muscle fiber morphometry. [¹⁸F]DPA-714 uptake was significantly increased in distal hindlimb of crush-injured mice compared with contralateral and sham models. PET signal was selectively confined to denervated muscle distal to the lesion. Tracer uptake ratio between injured and contralateral hindlimb clearly discriminated crush-injured from sham animals without overlap. Immunofluorescence demonstrated marked macrophage infiltration, Western blot confirmed increased TSPO protein levels, and laminin morphometry revealed reduced muscle fiber size consistent with early atrophic remodeling.

Conclusions

TSPO-targeted PET with [¹⁸F]DPA-714 enables an early and anatomically selective detection of denervation-induced skeletal muscle injury distal to the nerve lesion, supporting its role as a translational imaging biomarker of peripheral nerve-muscle interaction during acute reversible nerve damage.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s13550-026-01497-7.

Introduction

Peripheral nerve injury induces biological responses that extend beyond the site of axonal damage, leading to functional denervation and remodeling of distal skeletal muscles [1, 2]. In traumatic neuropathies, muscle represents the final effector of neural dysfunction and a major determinant of clinical disability [3]. Current diagnostic evaluation primarily focuses on electrophysiology and structural imaging of the affected nerve, whereas downstream skeletal muscle involvement remains largely unexplored. Nevertheless, identifying biological alterations in denervated muscle might provide complementary information during the initial phase of nerve injury.

An early hallmark of denervation is the development of an inflammatory response within affected muscle territories [4, 5]. Following axonal disruption, immune cell infiltration—particularly CD68⁺ macrophages—occurs in distal muscle before overt structural atrophy becomes evident [6, 7]. This spatially confined remodeling reflects the downstream biological impact of neural injury and may define a temporally restricted window suitable for molecular imaging.

The 18-kDa translocator protein (TSPO) is a mitochondrial outer membrane protein upregulated in activated macrophages and in cells exposed to inflammatory and metabolic stress [8–11]. Because TSPO expression reflects the integrated inflammatory-metabolic state of tissue, PET imaging of TSPO enables non-invasive visualization of stress-related biological activity in vivo. Among second-generation TSPO radioligands, [¹⁸F]DPA-714 demonstrates high affinity, favorable pharmacokinetics, and improved signal-to-noise ratio compared with first-generation tracers [12–14]. While its clinical application has primarily focused on central neuroinflammatory disorders [15–18], TSPO PET has also been shown to detect inflammatory activity in peripheral tissues, including myocardium and vascular structures [19–22], justifying its potential extension to skeletal muscle injury [23–25].

The sciatic nerve crush represents a well-established model of transient peripheral nerve injury characterized by reversible axonal degeneration followed by progressive reinnervation of distal target muscles [26]. During the early post-injury phase, denervated muscle exhibits pronounced inflammatory activation [5], resembling early stages of reversible nerve damage observed in clinical conditions such as compressive or traumatic neuropathies [27].

In this study, we investigated whether [¹⁸F]DPA-714 PET enables non-invasive detection and distal localization of early inflammatory remodeling in skeletal muscle following sciatic nerve crush. We aimed to assess the feasibility of TSPO PET as a translational imaging biomarker of nerve-muscle interaction during the initial phase of reversible peripheral nerve injury.

Materials and methods

Radiochemistry

[¹⁸F]DPA-714 was synthesized on-site according to the procedure previously described [13, 23], using an optimized fully automated method on a Trasis AllinOne module (Trasis, Ans, Belgium). [¹⁸F]Fluoride was reacted with the tosylated DPA-714 precursor through nucleophilic substitution in anhydrous acetonitrile. The final product [¹⁸F]DPA-714 was purified by semi-preparative HPLC, followed by sterile filtration.

The total synthesis time was approximately 45 min. Radiochemical purity exceeded 98%, and molar activity was > 150 GBq/μmol at the end of synthesis. All reagents were of pharmaceutical grade, and quality control was performed in compliance with European Pharmacopoeia and ICH Q2(R1) guidelines.

Animals and experimental design

Adult male C57BL/6 J mice (3.5–5.5-month-old; 25–30 g) were obtained from Charles River Laboratories Italia s.r.l. (Milan, Italy) and housed under controlled environmental conditions (12:12 h light/dark cycle, 22 ± 2 °C, 55 ± 10% humidity) with food and water provided ad libitum in the Animal Facility of our Institution.

All experimental procedures were performed in accordance with the Italian Legislative Decree 26/2014 and the EU Directive 2010/63/EU for the care and use of laboratory animals. The protocol was approved by the Institutional Animal Welfare Body (OPBA) and authorized by the Italian Ministry of Health (project number approval 301/2023-PR, 2023–04-11).

Mice were randomly assigned to one of the following experimental groups: (i) Crush (n = 7), evaluated seven days after sciatic nerve crush injury; and (ii) Sham (n = 6), subjected to surgical exposure of the sciatic nerve without compression and used as time-matched controls. Contralateral uninjured limbs from both groups served as internal controls.

Sciatic nerve crush injury

Sciatic nerve crush injury was performed as previously described [28]. Mice were anesthetized with isoflurane (4% induction, 2% maintenance in oxygen), a skin incision was made at the right sciatic notch, and the gluteal muscles were bluntly separated to expose the sciatic nerve. After gentle isolation from surrounding connective tissue, the nerve was crushed at the sciatic notch using Dumont No. 5 forceps (7 mm), previously cooled in liquid nitrogen.

The crush was applied three times for 15 s, a protocol known to induce complete axonal degeneration while allowing subsequent regeneration. Sham-operated mice underwent the same surgical exposure without nerve compression. The muscle and skin were closed with interrupted sutures. Lidocaine 2% (50 µL) was applied locally, and diclofenac (10 mg/kg, intraperitoneal) was administered in all mice once immediately after surgery for postoperative analgesia, without additional administrations on the following days. Contralateral uninjured limbs from both crush and sham animals served as internal controls. PET imaging was performed one week after surgery.

Small-animal PET Imaging

PET imaging was performed using a dedicated small-animal PET scanner (Albira, Bruker, USA). Mice were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg, intraperitoneal) and positioned prone with hindlimbs extended. Each animal received an intravenous injection of [¹⁸F]DPA-714 (7–10 MBq) via the tail vein. A static PET acquisition was started 30 min post-injection and lasted 10 min, covering both hindlimbs. The static acquisition window was selected based on previous preclinical experience with [¹⁸F]DPA-714 imaging in mouse skeletal muscle [23]. PET data were reconstructed using a maximum likelihood expectation maximization (MLEM) algorithm and corrected for radioactive decay.

Image analysis

Reconstructed PET images were analyzed using PMOD software (version 3.405, PMOD Technologies, Zurich, Switzerland) by two independent observers unaware of the experimental model, and the average value was considered. For each mouse, tracer uptake was quantified in the distal hindlimb skeletal muscle by the manual definition of volumes of interest (VOIs) considering ≥ 3 consecutive PET slices to cover the distal hindlimb. VOIs were positioned distally to the surgical site and carefully drawn to include the visible muscle compartment while minimizing the contribution of adjacent non-muscular structures. Care was taken to avoid skin contamination and regions potentially affected by urinary spillover.

Because hindlimb size varied across animals, VOI size was anatomically adapted to each mouse rather than kept fixed across the cohort. VOI placement was reviewed in axial, coronal, and sagittal planes. VOI volumes were checked to confirm side-to-side comparability within each animal.

Tracer uptake was expressed as standardized uptake value (SUV), calculated as:

The mean SUV (SUV-mean) was calculated for each VOI. In crush-injured mice, uptake was compared between the injured and contralateral limbs, whereas in sham-operated mice right-to-left uptake symmetry was assessed. In addition, an injured-to-contralateral SUV ratio was calculated for crush animals and compared with the corresponding right-to-left ratio in sham-operated controls.

Tissue collection

At 24 h after image acquisition, mice fulfilling humane endpoint criteria, defined as failure to recover the righting reflex within 30 s in lateral recumbency, were euthanized by CO₂ inhalation. Tibialis anterior muscles were promptly collected for downstream analyses. In one animal, harvested specimen was of inadequate quality. Therefore, histological analyses were performed on 6 of the 7 animals.

Hematoxylin and eosin staining

Tibialis anterior muscles were fixed in 10% neutral buffered formalin for 24 h at 4 °C. After routine paraffin embedding using an automated tissue processor, specimens from the three experimental groups were cut into 4-µm-thick sections using a rotary microtome (Leica RM 2135, Leica Biosystems, Milan, Italy). Sections were mounted on glass slides and stained with hematoxylin and eosin according to standard protocols [23].

Immunofluorescence

To evaluate muscle fiber size, inflammatory infiltrate, and TSPO localization, OCT-embedded tibialis anterior muscles from each experimental group (Control, Sham and Crush, n = 6/group) were cryosectioned at 10 µm thickness, mounted on Superfrost adhesive glass slides (Epredia, J1800AMNZ), and stored at -80 °C until processing.

Frozen sections were rinsed in Dulbecco’s phosphate-buffered saline (DPBS; Thermo Fisher Scientific, 14,190–144), fixed in 4% paraformaldehyde for 20 min, and permeabilized and blocked for 30 min in buffer containing 15% normal goat serum, 0.3% Triton X-100, 20 mM phosphate buffer, and 450 mM NaCl. Sections were then incubated overnight at 4 °C with primary antibodies against laminin α-2 (Santa Cruz Biotechnology, sc-59854, 1:200), TSPO (Thermo Fisher Scientific, MA5-24,844, 1:200), and CD68 (Bio-Rad, MCA1957, 1:200).

After washing in DPBS, sections were incubated for 90 min at room temperature with Alexa Fluor 488-conjugated goat anti-rabbit or Alexa Fluor 594-conjugated goat anti-rat IgG secondary antibodies (Thermo Fisher Scientific, A11007, A11008; 1:400). Nuclei were counterstained with DAPI (300 nM), and sections were mounted using Fluoromount aqueous mounting medium.

Whole-section images were acquired at 10 × magnification using an Olympus PROVIS AX60 microscope equipped with an Olympus DP74 digital camera. Image analysis was performed using Fiji [29]. TSPO- and CD68-positive areas were quantified in longitudinal tibialis anterior muscle sections by applying a uniform threshold across all images and calculating the percentage area occupied by positive signal. Myofiber size was assessed in laminin α-2–stained tibialis anterior muscle cross-sections using the Open-CSAM plugin [30], to estimate the cross-sectional area and the minimal Feret’s diameter (MFD), defined as the shortest distance between two parallel tangents to the fiber boundary [31].

TSPO- and CD68-positive areas were quantified in tibialis anterior muscle sections by applying a uniform threshold across all images and calculating the percentage area occupied by positive signal. Colocalization of TSPO and CD68 fluorescent signals was evaluated using the Coloc 2 plugin of ImageJ (https://imagej.net/plugins/coloc-2), using point spread function = 3 and Costes randomization = 10 [32, 33].

Western blot analysis

Tibialis anterior muscles collected from uninjured control, sham, and crush mice seven days after surgery were homogenized in RIPA lysis buffer (150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris–HCl, pH 7.5) supplemented with NaF (5 mM), NaVO₄ (1 mM), phenylmethylsulfonyl fluoride (PMSF, 1 mM), and a protease and phosphatase inhibitor cocktail (Merck, P8340; 1:100).

Protein concentration was determined using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, 23,227) according to the manufacturer’s instructions. Equal amounts of proteins (25 µg per sample) were separated by SDS-PAGE on 4–15% Mini-PROTEAN® TGX Stain-Free™ gels (Bio-Rad, 4,568,085). Gels were activated and imaged using a Bio-Rad ChemiDoc™ MP Imaging System to enable total protein normalization, then transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, 1,620,177).

After transfer, membranes were imaged to quantify total protein per lane and subsequently blocked for 1 h at room temperature in 5% bovine serum albumin (BSA) in Tris-buffered saline containing 0.1% Tween-20 (TBST). Membranes were incubated overnight at 4 °C with an anti-TSPO primary antibody (1:1000). After washing with TBST, membranes were incubated for 90 min at room temperature with HRP-conjugated secondary antibody (Santa Cruz Biotechnology, sc-2357; 1:5000).

Immunoreactive bands were visualized using Clarity™ Western ECL Substrate (Bio-Rad, 1,705,060) and imaged with the ChemiDoc™ MP Imaging System. Densitometric analysis was performed using Image Lab software (version 6.1.0, Bio-Rad).

Statistical analysis

Data are presented as mean ± standard deviation. Data distribution was assessed using the Shapiro–Wilk normality test. For two-group comparisons, paired or unpaired Student’s t-tests were used, as appropriate, when normality was not rejected. When normality was rejected (p < 0.05), the corresponding non-parametric test was applied (Wilcoxon signed-rank test for paired data or Mann–Whitney U test for unpaired data). For comparisons among more than two groups, one-way analysis of variance (ANOVA) followed by Tukey’s multiple-comparison test was used when normality was not rejected in all groups. When normality was rejected in at least one group, the Kruskal–Wallis test followed by Dunn’s post hoc test was applied. For the colocalization analysis, significance was calculated using Costes’s significance test [33]. Correlations between PET and ex vivo measurements were also assessed. To minimize inter-experimental variability, data from each experiment were normalized and expressed as a percentage of the mean value of the corresponding experimental condition. Associations between variables were assessed using Pearson’s correlation coefficient and visualized by linear regression analysis. Spearman’s rank correlation was additionally performed to confirm the robustness of the findings. Statistical analyses were performed using GraphPad Prism version 8 (GraphPad Software, San Diego, CA, USA), with significance set at p < 0.05.

Results

[¹⁸F]DPA-714 PET reveals increased TSPO signal in functionally denervated skeletal muscle following sciatic nerve injury

Body weight of studied mice was not altered by the nerve injury (35.7 ± 4.4 g vs 35.6 ± 3.6 in sham and injured mice, respectively, unpaired Student’s t-test, p = 0.97). Static [¹⁸F]DPA-714 PET imaging at 30 min post-injection showed symmetrical uptake at the thigh level. In contrast, and consistent with the anatomical territory of the sciatic nerve, tracer uptake was selectively increased in the distal portion of the denervated right hindlimb, with values significantly higher than those of the contralateral limb (paired Student’s t-test, p < 0.001; Fig. 1A-B).

Fig. 1.

Increased [¹⁸F]DPA-714 uptake in distal hindlimb muscles one-week after sciatic nerve crush. (A) Representative maximum-intensity projection PET images acquired 30 min after intravenous injection of [¹⁸F]DPA-714 in mice one-week after sciatic nerve crush (Crush) and in sham-operated controls (Sham). Increased tracer uptake is evident in distal hindlimb muscles of the injured limb (red arrows), whereas tracer distribution appears symmetric in sham animals. The color scale represents normalized standardized uptake values (SUV; range 0–1). (B) Quantitative analysis of tracer uptake (SUV-mean) in distal hindlimb muscles. In crush-injured mice, SUV-mean was significantly higher in the injured limb compared with the contralateral side, reported as control (*** = p < 0.001 at paired Student’s t test,). No significant right-to-left difference was observed in sham animals (paired Student’s t test, n.s.). Each dot represents one animal. (C) Hindlimb uptake ratio analysis (injured-to-control for Crush; right-to-left for Sham). The ratio was significantly higher in crush-injured mice than in sham ones (***p = 0.001 at unpaired Student’s t test). Each dot represents one animal, bars indicate mean values

In sham animals, tracer uptake was superimposable in right and left hindlimb (paired Student’s t-test, not significant; Fig. 1A-B), indicating preserved uptake symmetry. In contrast, crush-injured mice exhibited a significant asymmetry between injured and contralateral limbs (injured-to-contralateral ratio; unpaired Student’s t-test, p = 0.001; Fig. 1C). Notably, tracer uptake in the left – not damaged – hindlimb was not significantly different in the two cohorts (0.28 ± 0.08 vs 0.31 ± 0.07, respectively, p = 0.4). By contrast the injured-to-contralateral SUV ratio did not overlap with that observed in sham mice, further supporting asymmetric [¹⁸F]DPA-714 uptake as a quantitative indicator of denervation-related inflammatory activation in distal skeletal muscle.

Ex vivo validation of muscle inflammation and TSPO upregulation in denervated skeletal muscle

Histological examination of hematoxylin and eosin–stained tibialis anterior sections collected one-week after sciatic nerve crush revealed a clear inflammatory infiltrate in the ipsilateral, denervated muscle (Fig. 2A). Inflammatory cell accumulation was evident within interstitial spaces and around vascular structures, whereas no comparable alterations were observed in contralateral or sham-operated muscles.

Fig. 2.

Ex vivo validation of inflammation and TSPO upregulation in denervated skeletal muscle. Representative hematoxylin and eosin (H&E)—stained sections (A) and CD68 immunofluorescence images (B) of tibialis anterior muscle collected one-week after surgery from contralateral hindlimb (Control), sham-operated (Sham), and homolateral hindlimb of injured sciatic nerve (Crush). CD68-positive macrophages are shown in red; nuclei are counterstained with DAPI (blue). Scale bar: 50 µm. (C) Quantification of CD68-positive area, expressed as percentage of total tissue area. Transiently denervated muscles showed significantly greater macrophage infiltration than control and sham groups. Each dot represents one biological replicate (one muscle per animal). Statistical analysis was performed using Kruskal–Wallis test, followed by Dunn’s post hoc test (*p < 0.05; **p < 0.01)

Immunofluorescence analysis confirmed macrophage recruitment. CD68 staining demonstrated a marked increase in macrophage-positive cells in denervated muscle compared with both control and sham groups (Fig. 2B). Quantitative analysis showed a significantly higher CD68-positive area in muscles downstream of the crushed nerve (Fig. 2C).

Consistent with the PET findings, TSPO immunostaining revealed increased TSPO expression in muscles distal to the crushed nerve compared with contralateral uninjured limbs and sham-operated mice (Fig. 3A). Quantitative analysis revealed a significantly greater TSPO-positive area in muscles from crush-injured hindlimbs. (Fig. 3B). Immunofluorescence analysis of TSPO and CD68 displayed a high degree of colocalization of the two markers (Pearson’s correlation coefficient = 0.55 ± 0.03; Costes randomization P-value = 1 ± 0.01), indicating that TSPO expression is predominantly associated with CD68-positive cells (Fig. 3C). To further corroborate the imaging findings at the molecular level, TSPO protein expression was assessed by Western blot. TSPO levels were significantly elevated in the ipsilateral tibialis anterior muscle compared with control and sham muscles, which displayed comparable expression levels (Fig. 3D-E, original gels reported as Suppl Fig. 1).

Fig. 3.

Early structural remodeling and onset of denervation-induced muscle atrophy following sciatic nerve injury. (A) Representative immunofluorescence images of TSPO (green) signal in skeletal muscle cross-sections from tibialis muscle harvested from contralateral hindlimb (Control), sham-operated (Sham), and homolateral side of injured sciatic nerve crush (Crush). Nuclei are counterstained with DAPI (blue). Specimen downstream the nerve damage showed a marked increase in % area occupied by TSPO immunofluorescence (B). CD68 and TSPO immunostaining and their colocalization in the tibialis anterior muscle downstream the nerve damage. (D) Representative Western blot analysis of TSPO protein levels in tibialis anterior muscle homogenates one-week after surgery. Total protein staining was used for normalization. (E) Densitometric quantification of TSPO expression normalized to total protein. TSPO levels were significantly increased in denervated muscle compared with control and sham groups, whereas no difference was observed between control and sham mice. Data are presented as mean ± SD; each dot represents one biological replicate. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test (***p < 0.001)

Overall, the increase in TSPO expression and macrophage infiltration occurred in the same muscle exhibiting enhanced [¹⁸F]DPA-714 uptake, supporting a tissue-level association between TSPO upregulation, inflammatory remodeling, and the PET signal observed in vivo.

Early structural remodeling and onset of denervation-induced muscle atrophy

Considering the inflammatory and molecular changes observed, we next investigated whether structural alterations were already detectable at this early time point. Laminin staining of muscle cross-sections revealed clear evidence of structural remodeling in the muscle downstream of the crushed nerve at one-week post-injury (Fig. 4A). Quantitative analysis of MFD demonstrated that nerve damage significantly decreased the average size (Fig. 4B). Moreover, assessing the distribution of myocyte size showed that skeletal muscles downstream the nerve damage were characterized by an increased prevalence of fibers with relatively low cross-sectional area as opposed to a relative lessening of larger ones, suggesting an early onset of denervation-induced muscle atrophy (Fig. 4C).

Fig. 4.

Early structural remodeling and onset of denervation-induced muscle atrophy following sciatic nerve injury. (A) Representative immunofluorescence images of skeletal muscle cross-sections collected one week after surgery from tibialis muscle harvested from contralateral hindlimb (Control), sham-operated (Sham), and homolateral side of injured sciatic nerve crush (Crush). Sections were stained for laminin (LAM, green) to delineate myofiber boundaries and counterstained with DAPI (blue) to label nuclei. Scale bar: 50 µm. (B) Histogram of minimal Feret’s diameter documenting the significant reduction in fiber caliber in denervated muscles compared with control and sham-operated ones. Individual data points represent one biological replicate. (C) Distribution of cross-sectional areas showing the increased prevalence of small fibers paralleled by an increased prevalence of large ones in tibialis anterior muscle downstream the nerve damage. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test (**p < 0.01)

These structural alterations were observed in the same muscle displaying increased TSPO expression and macrophage infiltration, suggesting that inflammatory and mitochondrial stress responses accompany early denervation-induced atrophy.

In vivo [18F]DPA-714 uptake correlates with ex vivo markers of muscle inflammation and remodeling

To further assess whether the in vivo PET signal reflected tissue-level inflammatory and remodeling changes, we performed correlation analyses between muscle [18F]DPA-714 uptake and the corresponding ex vivo markers across individual muscle samples. To minimize inter-experimental variability, data from each experiment were normalized and expressed as a percentage of the mean value of the corresponding experiment. In all cases, Pearson correlation results were confirmed by Spearman rank correlation analysis, which yielded comparable results.

[18F]DPA-714 uptake positively correlated with both CD68 and TSPO immunofluorescence, supporting an association between tracer accumulation, macrophage/inflammatory cell infiltration, and tissue TSPO expression (Fig. 5A-B). This observation was further strengthened by the direct correlation between tracer uptake and TSPO protein expression assessed by Western blot (Fig. 5C). Finally, [18F]DPA-714 uptake was also positively correlated with minimum Feret diameter (Fig. 5D), suggesting a relationship between PET signal and muscle structural changes. Collectively, these findings support the ability of [18F]DPA-714 PET to capture biologically relevant inflammatory processes occurring in denervated skeletal muscle.

Fig. 5.

Correlation between in vivo [18F]DPA-714 uptake and ex vivo markers of muscle inflammation and remodeling. Scatter plots showing the association between muscle [18F]DPA-714 uptake, expressed as SUV, and corresponding ex vivo tissue markers: CD68 immunoreactivity (A), TSPO immunofluorescence (B), TSPO protein expression by Western blot (C), and minimum Feret diameter (MFD) as an index of myofiber size (D). Each point represents an individual muscle sample normalized as % of the mean value observed in the corresponding experimental group. Injured/crush, contralateral/control, and sham muscles are shown with distinct colors. Solid lines indicate the best-fit linear regression. R² and p values are reported in each panel

Discussion

The present study provides proof-of-concept evidence that TSPO-targeted PET enables non-invasive and anatomically selective detection of early inflammatory remodeling in skeletal muscle following transient peripheral nerve injury. Using the sciatic nerve crush model—a well-established paradigm of a peripheral nerve trauma—we demonstrate that [¹⁸F]DPA-714 uptake selectively identifies the denervated muscle distal to the lesion during the initial biological response to nerve damage. Axonal disruption rapidly triggers an inflammatory response within the distal target muscle eventually resulting in macrophage recruitment with stress-related molecular adaptation emerging within days after injury and preceding overt structural degeneration [5, 6, 26]. In vivo PET findings closely paralleled these events: tracer uptake was selectively increased in muscle innervated by the crushed nerve, while remaining unchanged in proximal thigh. The absence of cohort overlap in the injured-to-contralateral uptake ratio supports the robustness of TSPO PET as a semi-quantitative imaging readout of denervation-associated inflammatory activation. Importantly, the most robust imaging finding was not the absolute SUV value per se, but the emergence of a selective side-to-side uptake asymmetry involving the distal hindlimb after sciatic nerve crush. This paired analysis reduces the impact of inter-animal variability and supports the interpretation of [¹⁸F]DPA-714 PET as a spatial readout of denervation-associated tissue remodeling rather than as an absolute measure of TSPO binding density.

Ex vivo analyses further supported the biological interpretation of the PET signal. Hematoxylin and eosin staining demonstrated inflammatory cell infiltration, confirmed by prominent CD68⁺ macrophage accumulation on immunofluorescence. Western blot analysis showed an increased TSPO protein expression in the same tissue. Laminin-based morphometry revealed reduced muscle fiber size at this early time point, consistent with incipient atrophic remodeling. Together, these findings indicate that increased tracer uptake occurs during a phase characterized by active inflammatory and stress responses preceding overt structural degeneration [1, 6, 7, 26].

Although activated macrophages likely represent a major source of tracer accumulation, stressed myofibers may also contribute, consistent with the role of TSPO as an indicator of mitochondrial dysfunction and cellular stress rather than a strictly cell-type specific marker. This integrated cellular contribution supports the interpretation of TSPO PET as a readout of the combined inflammatory-metabolic response of the denervated muscle microenvironment.

The sciatic nerve crush model recapitulates clinically relevant scenarios of transient and potentially reversible peripheral nerve damage, analogous to compressive neuropathies and acute traumatic injuries [34]. Structural nerve imaging techniques including high-resolution MR neurography, diffusion tensor imaging, and high-frequency ultrasound [35–38] provide essential information on nerve integrity and regeneration but primarily characterize the injured nerve itself. By visualizing inflammation and stress responses within muscle distal to the lesion, TSPO PET offers complementary information on downstream tissue involvement during the acute injury phase.

From a translational perspective, imaging biomarkers capable of capturing early peripheral tissue responses are increasingly recognized as critical tools for disease monitoring and therapeutic stratification [39–41]. Early detection of muscle involvement may support patient selection, optimize intervention timing, and potentially serve as a surrogate marker of treatment response. Beyond identifying inflammation, TSPO PET may provide insight into nerve–muscle interaction during the acute phase of injury and, in future longitudinal studies, throughout regeneration [25, 34].

While these findings support the feasibility and translational relevance of TSPO PET in this setting, certain methodological considerations should be acknowledged.

Limitations

Several limitations warrant consideration. Analyses were restricted to a single early post-injury time point selected to capture acute inflammatory activation; therefore, regenerative and reinnervation dynamics were not longitudinally assessed. Future studies incorporating serial imaging will be necessary to define the temporal evolution of TSPO expression during muscle recovery.

As a second point, PET acquisitions were performed using a static protocol, without kinetic modeling and without a validated skeletal-muscle reference region for [¹⁸F]DPA-714. This procedure was selected to obtain a reproducible semi-quantitative readout of distal hindlimb tracer accumulation while limiting acquisition time and anesthesia duration in small animals. This time schedule was based on previous studies documenting that the increase tracer concentration markedly decelerates 30 min after injection both in mice [23, 25] and humans [42]. Actually, this analysis reported a relevant variability in tracer uptake in the left, not injured, hindlimb. However average SUV of this district was not significantly different in crush mice with respect to sham ones.

As a further limitation of our imaging approach, PET scans were not complemented with co-registered CT or MRI images. Although providing reproducible anatomically matched distal hindlimb measurements, obtained SUV values should be interpreted as semi-quantitative indices of tracer accumulation rather than direct estimates of TSPO binding density.

Nevertheless, the nerve damage markedly increased the uptake ratio between injured and contralateral side up to the absence of any overlap with respect to sham mouse, suggesting that the evident asymmetry might represent a promising diagnostic index potentially able to identify the nerve damage.

A further limitation is the exclusive use of male mice. This design reduced sex-related biological variability within the experimental cohort, but limits generalizability to both sexes. Although previous evidence indicates comparable axonal regeneration after sciatic nerve crush in male and female mice [34], future studies including female animals will be needed to confirm whether TSPO-related inflammatory responses after denervation injury are similarly represented in both sexes.

Finally, while imaging and molecular findings support an inflammatory basis of the TSPO signal, the relative contribution of distinct cellular compartments requires further clarification through cell-type–resolved investigations.

Conclusion

TSPO-targeted PET imaging with [¹⁸F]DPA-714 provides a sensitive and non-invasive approach to detect skeletal muscle involvement following peripheral nerve injury. By capturing inflammation-related tissue responses in vivo, this strategy enables anatomically selective identification of muscle changes distal to the site of neural damage and extends the application of TSPO imaging beyond central neuroinflammation to peripheral neuromuscular pathology.

These findings position TSPO PET as a promising translational imaging biomarker for assessing early muscle response to peripheral nerve injury and related neuropathies.

Author contributions

All authors contributed to the study conception and design. SL, IdP, MG, MR, RR, SC, MC, GC, FV, and SC were involved in material preparation was performed by. Data collection was performed by ED, LE, RR, AMO and LN. Data analysis was performed by SL, ED, MB, LS, RP, MH, FZ, CaM, AS, GS and CeM. The first draft of the manuscript was written by SL and CeM. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding

The study has been funded by the Italian Ministry of Health in the context of the project T4-AN-10 (Hybrid Hub (H2UB): Modelli cellulari e computazionali, micro e nanotecnologie per la personalizzazione di terapie innovative), by the Italian Ministry of Research in the context of the program PE 0000006, MNESYS—A multiscale integrated approach to the study of the nervous system in health and disease, and, finally, by the Italian Ministry of Health in the context of the program “Ricerca Finalizzata 2021” and the number RF-2021–12372711.

Data availability

The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

All experimental procedures were performed in accordance with the Italian Legislative Decree 26/2014 and the EU Directive 2010/63/EU for the care and use of laboratory animals. The protocol was approved by the Institutional Animal Welfare Body (OPBA) and authorized by the Italian Ministry of Health (project number approval 301/2023-PR, 2023–04-11). The study only involved experimental animals.

Consent for publication

The study only involved experimental animals.

Competing interests

The authors have no relevant financial or non-financial interests to disclose.