The Masquelet Technique: Can Disposable Polypropylene Syringes be an Alternative to Standard PMMA Spacers? A Rat Bone Defect Model.

BACKGROUND: Usually, the two-stage Masquelet induced-membrane technique for extremity reconstruction begins with a polymethylmethacrylate (PMMA) cement spacer-driven membrane, followed by an autologous cancellous bone graft implanted into the membrane cavity to promote healing of large bone defects. In exceptional cases, spacers made of polypropylene disposable syringes were successfully used instead of the usual PMMA spacers because of a PMMA cement shortage caused by a lack of resources. However, this approach lacks clinical evidence and requires experimental validation before being recommended as an alternative to the conventional technique. QUESTIONS/PURPOSES: To (1) develop and (2) validate a critical-sized femoral defect model in rats for two stages of the Masquelet technique and to (3) compare the biological and bone healing properties of polypropylene-induced membranes and PMMA-induced membranes in this model. METHODS: Fifty male Sprague Dawley rats aged 8 weeks old received a 6-mm femur defect, which was stabilized with an external fixator that was converted into an internal device. In the development phase, the defect was filled with PMMA in 16 rats to determine the most favorable timing for bone grafting. Two rats were excluded since they died of anesthetic complications. The other 14 were successively euthanized after 2 weeks (n = 3), 4 weeks (n = 4), 6 weeks (n = 4), and 8 weeks (n = 3) for induced membrane analyses. In the validation phase, 12 rats underwent both stages of the procedure using a PMMA spacer and were randomly assigned to two groups, whether the induced membrane was preserved or removed before grafting. To address our final objective, we implanted either polypropylene or PMMA spacers into the defect (Masquelet technique Stage 1; n = 11 rats per group) for the period established by the development phase. In each group, 6 of 11 rats were euthanized to compare the biological properties of polypropylene-induced membranes and PMMA-induced membranes using histological qualitative analysis, semiquantitative assessment of the bone morphogenic protein-2 content by immunostaining, and qualitative assessment of the mesenchymal stromal cell (MSC; CD31-, CD45-, CD90+, and CD73+ phenotypes) content by flow cytometry. Quantitative measurements from serum bone turnover markers were also performed. The five remaining rats of each group were used for Masquelet technique Stage 2, in which rat bone allografts were implanted in the induced membrane cavity after the polypropylene or PMMA spacers were removed. These rats recovered for 10 weeks before being euthanized for microCT quantitative measurements and bone histology qualitative assessment to evaluate and compare the extent of bone regeneration between groups. RESULTS: Induced membrane analyses together with serum bone turnover measurements indicated that a 4-week interval time between stages was the most favorable. Removal of the induced membrane before grafting led to almost constant early implant failures with poor bone formation. Four-week-old rats with polypropylene-triggered induced membranes displayed similar histologic organization as rats with PMMA-driven induced membranes, without any difference in the cell density of the extracellular matrix (4933 ± 916 cells per mm2 for polypropylene versus 4923 ± 1284 cells per mm2 for PMMA; p = 0.98). Induced membrane-derived MSCs were found in both groups with no difference (4 of 5 with polypropylene versus 3 of 3 with PMMA; p > 0.99). Induced membrane bone morphogenic protein-2 immunolabeling and serum bone turnover marker levels were comparable between the polypropylene and PMMA groups. MicroCT analysis found that bone regeneration in the polypropylene group seemed comparable with that in the PMMA group (29 ± 26 mm3 for polypropylene versus 24 ± 18 mm3 for PMMA; p > 0.99). Finally, qualitative histological assessment revealed a satisfactory endochondral ossification maturation in both groups. CONCLUSION: Using a critical-sized femoral defect model in rats, we demonstrated that polypropylene spacers could induce membrane encapsulation with histologic characteristics and bone regenerative capacities that seem like those of PMMA spacers. CLINICAL RELEVANCE: In a same bone site, polymers with close physical properties seem to lead to similar foreign body reactions and induce encapsulating membranes with comparable bone healing properties. Polypropylene spacers made from disposable syringes could be a valuable alternative to PMMA. These results support the possibility of a cementless Masquelet technique in cases of PMMA shortage caused by a lack of resources.

Calcium-phosphate ceramics and polysaccharide-based hydrogel scaffolds combined with mesenchymal stem cell differently support bone repair in rats.

Research in bone tissue engineering is focused on the development of alternatives to autologous bone grafts for bone reconstruction. Although multiple stem cell-based products and biomaterials are currently being investigated, comparative studies are rarely achieved to evaluate the most appropriate approach in this context. Here, we aimed to compare different clinically relevant bone tissue engineering methods and evaluated the kinetic repair and the bone healing efficiency supported by mesenchymal stem cells and two different biomaterials, a new hydrogel scaffold and a commercial hydroxyapatite/tricalcium phosphate ceramic, alone or in combination.Syngeneic mesenchymal stem cells (5 × 105) and macroporous biphasic calcium phosphate ceramic granules (Calciresorb C35®, Ceraver) or porous pullulan/dextran-based hydrogel scaffold were implanted alone or combined in a drilled-hole bone defect in rats. Using quantitative microtomography measurements and qualitative histological examinations, their osteogenic properties were evaluated 7, 30, and 90 days after implantation. Three months after surgery, only minimal repair was evidenced in control rats while newly mineralized bone was massively observed in animals treated with either hydrogels (bone volume/tissue volume = 20%) or ceramics (bone volume/tissue volume = 26%). Repair mechanism and resorption kinetics were strikingly different: rapidly-resorbed hydrogels induced a dense bone mineralization from the edges of the defect while ceramics triggered newly woven bone formation in close contact with the ceramic surface that remained unresorbed. Delivery of mesenchymal stem cells in combination with these biomaterials enhanced both bone healing (>20%) and neovascularization after 1 month, mainly in hydrogel.Osteogenic and angiogenic properties combined with rapid resorption make hydrogels a promising alternative to ceramics for bone repair by cell therapy.

Masquelet technique in military practice: specificities and future directions for combat-related bone defect reconstruction.

Because of its simplicity, reliability, and replicability, the Masquelet induced membrane technique (IMT) has become one of the preferred methods for critical bone defect reconstruction in extremities. Although it is now used worldwide, few studies have been published about IMT in military practice. Bone reconstruction is particularly challenging in this context of care due to extensive soft-tissue injury, early wound infection, and even delayed management in austere conditions. Based on our clinical expertise, recent research, and a literature analysis, this narrative review provides an overview of the IMT application to combat-related bone defects. It presents technical specificities and future developments aiming to optimize IMT outcomes, including for the management of massive multi-tissue defects or bone reconstruction performed in the field with limited resources.

Macrophages Characterization in an Injured Bone Tissue.

Biomaterial use is a promising approach to facilitate wound healing of the bone tissue. Biomaterials induce the formation of membrane capsules and the recruitment of different types of macrophages. Macrophages are immune cells that produce diverse combinations of cytokines playing an important role in bone healing and regeneration, but the exact mechanism remains to be studied. Our work aimed to identify in vivo macrophages in the Masquelet induced membrane in a rat model. Most of the macrophages in the damaged area were M2-like, with smaller numbers of M1-like macrophages. In addition, high expression of IL-1ß and IL-6 cytokines were detected in the membrane region by RT-qPCR. Using an innovative combination of two hybridization techniques (in situ hybridization and in situ hybridization chain reaction (in situ HCR)), M2b-like macrophages were identified for the first time in cryosections of non-decalcified bone. Our work has also demonstrated that microspectroscopical analysis is essential for macrophage characterization, as it allows the discrimination of fluorescence and autofluorescence. Finally, this work has revealed the limitations of immunolabelling and the potential of in situ HCR to provide valuable information for in vivo characterization of macrophages.

Influence of the Immune Microenvironment Provided by Implanted Biomaterials on the Biological Properties of Masquelet-Induced Membranes in Rats: Metakaolin as an Alternative Spacer.

Macrophages play a key role in the inflammatory phase of wound repair and foreign body reactions-two important processes in the Masquelet-induced membrane technique for extremity reconstruction. The macrophage response depends largely on the nature of the biomaterials implanted. However, little is known about the influence of the macrophage microenvironment on the osteogenic properties of the induced membrane or subsequent bone regeneration. We used metakaolin, an immunogenic material, as an alternative spacer to standard polymethylmethacrylate (PMMA) in a Masquelet model in rats. Four weeks after implantation, the PMMA- and metakaolin-induced membranes were harvested, and their osteogenic properties and macrophage microenvironments were investigated by histology, immunohistochemistry, mass spectroscopy and gene expression analysis. The metakaolin spacer induced membranes with higher levels of two potent pro-osteogenic factors, transforming growth factor-ß (TGF-ß) and bone morphogenic protein-2 (BMP-2). These alternative membranes thus had greater osteogenic activity, which was accompanied by a significant expansion of the total macrophage population, including both the M1-like and M2-like subtypes. Microcomputed tomographic analysis showed that metakaolin-induced membranes supported bone regeneration more effectively than PMMA-induced membranes through better callus properties (+58%), although this difference was not significant. This study provides the first evidence of the influence of the immune microenvironment on the osteogenic properties of the induced membranes.

Nerve agent intoxication: recent neuropathophysiological findings and subsequent impact on medical management prospects.

This manuscript provides a survey of research findings catered to the development of effective countermeasures against nerve agent poisoning over the past decade. New neuropathophysiological distinctive features as regards organophosphate (OP) intoxication are presented. Such leading neuropathophysiological features include recent data on nerve agent-induced neuropathology, related peripheral or central nervous system inflammation and subsequent angiogenesis process. Hence, leading countermeasures against OP exposure are down-listed in terms of pre-treatment, protection or decontamination and emergency treatments. The final chapter focuses on the description of the self-repair attempt encountered in lesioned rodent brains, up to 3months after soman poisoning. Indeed, an increased proliferation of neuronal progenitors was recently observed in injured brains of mice subjected to soman exposure. Subsequently, the latter experienced a neuronal regeneration in damaged brain regions such as the hippocampus and amygdala. The positive effect of a cytokine treatment on the neuronal regeneration and subsequent cognitive behavioral recovery are also discussed in this review. For the first time, brain cell therapy and neuronal regeneration are considered as a valuable contribution towards delayed treatment against OP intoxication. To date, efficient delayed treatment was lacking in the therapeutic resources administered to patients contaminated by nerve agents.

Induced membrane technique: a critical literature analysis and proposal for a failure classification scheme.

The reconstruction of long-bone segmental defects remains challenging, with the three common methods of treatment being bone transport, vascularized bone transfer, and the induced membrane technique (IMT). Because of its simplicity, replicability, and reliability, usage of IMT has spread all over the world in the last decade, with more than 300 papers published in the PubMed literature database on this subject so far. Most of the clinical studies have reported high rates of bone union, yet some also include more controversial results with frequent complications and revision surgeries. At the same time, various experimental research efforts have been designed to understand and improve the biological properties of the induced membrane. This literature review aims to provide an overview of IMT clinical results in terms of bone union and complications and to compare them with those of other reconstructive procedures. In light of our findings, we then propose an original classification scheme of IMT failures distinguishing between preventable and nonpreventable failures.

Repeated Induced-Membrane Technique Failure without Infection: A Series of Three Consecutive Procedures Performed for a Single Femur Defect.

A 40-year-old male was treated using the induced-membrane technique (IMT) for a noninfected, 9 cm long femoral bone defect complicating a lengthening procedure. The interesting case feature lies in the three consecutive IMT procedures that were necessary to achieve complete bone repair in this unusual clinical situation. The first procedure failed because of the lack of graft revascularization likely related to an induced-membrane (IM) alteration demonstrated by histological observations. The second IMT procedure led to partial graft integration interrupted by the elongation nail breakage. At last, the third procedure fully succeeded after nail exchange and iterative iliac bone grafting. Complete bone union was achieved with a poor functional recovery one year after the last procedure and four years following the first cement spacer implantation. By means of clinical and histological observations, we demonstrated that the first and the second IMT failures had two distinct origins, namely, biological and mechanical causes, respectively. Although simple, a successful IMT procedure is not so easy to complete.

Bone reconstruction by the induced membrane technique. What differences between conventional and ballistic trauma?

BACKGROUND: The induced membrane technique (IMT) has been widely evaluated for reconstruction of post-traumatic bone defects. However, no specific evaluation was conducted in ballistic injuries. The objective of the present study was to compare IMT in conventional trauma (CT) versus ballistic trauma (BT) managed in a military trauma center. METHODS: A retrospective study was conducted between 2009 and 2018 in patients treated by IMT for post-traumatic bone defects, whatever the defect location. Endpoints comprised bone union, residual infection, additional bone grafting and lower-limb amputation. RESULTS: Thirty-six patients were included: 24 in the CT and 12 in the BT group. Demographics and injury pattern were similar in both groups, with open fracture and infected lesions predominating. The only significant difference was that tibial bone defects were larger in the BT group. Operative parameters and results were also similar. At a mean 24 months' follow-up, bone union rate was 83% in both groups, without significant differences in residual infection, complementary grafting or late amputation. CONCLUSION: IMT is appropriate to bone reconstruction in the aftermath of ballistic trauma, with similar results to those obtained in conventional trauma. LEVEL OF EVIDENCE: IV, retrospective study.

Towards Understanding Therapeutic Failures in Masquelet Surgery: First Evidence that Defective Induced Membrane Properties are Associated with Clinical Failures.

The two-stage Masquelet induced-membrane technique (IMT) consists of cement spacer-driven membrane induction followed by an autologous cancellous bone implantation in this membrane to promote large bone defect repairs. For the first time, this study aims at correlating IMT failures with physiological alterations of the induced membrane (IM) in patients. For this purpose, we compared various histological, immunohistochemical and gene expression parameters obtained from IM collected in patients categorized lately as successfully (Responders; n = 8) or unsuccessfully (Non-responders; n = 3) treated with the Masquelet technique (6 month clinical and radiologic post-surgery follow-up). While angiogenesis or macrophage distribution pattern remained unmodified in non-responder IM as compared to responder IM, we evidenced an absence of mesenchymal stem cells and reduced density of fibroblast-like cells in non-responder IM. Furthermore, non-responder IM exhibited altered extracellular matrix (ECM) remodeling parameters such as a lower expression ratio of metalloproteinase-9 (MMP-9)/tissue inhibitor of metalloproteinases (TIMP-1) mRNA as well as an important collagen overexpression as shown by picrosirius red staining. In summary, this study is the first to report evidence that IMT failure can be related to defective IM properties while underlining the importance of ECM remodeling parameters, particularly the MMP-9/TIMP-1 gene expression ratio, as early predictive biomarkers of the IMT outcome regardless of the type of bone, fracture or patient characteristics.

Masquelet technique for open tibia fractures in a military setting.

PURPOSE: The induced membrane technique (IMT) is a two-stage procedure dedicated to reconstruction of bone defects of the limbs. The objective of this report was to evaluate employment of the IMT for the treatment of open tibia fractures managed in a military trauma center treating both wartime and peacetime injuries. METHODS: A retrospective study was performed among the patients treated via IMT for tibial bone defects related to open fractures between 2009 and 2018. The outcomes recorded included bone union, residual infection, amputation and lower limb function. RESULTS: During this period, 15 patients with a mean age of 39 years were included for the treatment of Gustilo II (2 cases) or Gustilo IIIB (13 cases) injuries. A mean number of 2.9 debridements were required before stage 1. Flap coverage was associated in 14 cases. The mean interval between stages was 22 weeks. Five patients were re-operated on after stage 1 due to persistent infection. The mean follow-up was 33 months. Bone union was achieved in 13 of the 15 cases (87%) at a mean time of 10.1 months. However, seven additional bone healing procedures were required, including six inter-tibiofibular grafting. Only one late septic recurrence was found. Most patients returned to work in sedentary jobs. CONCLUSIONS: This series is the first to report IMT use in a military setting. The prior eradication of infection constitutes a major challenge in tibial bone defects, especially in high-energy, multi-tissue injuries. An inter-tibiofibular bone reconstruction approach is required when external fixation is chosen.

Effects of soman poisoning on mitochondrial respiratory enzyme activity in the mouse hippocampus and cerebral cortex.

Mitochondrial dysfunctions have been highlighted as a contributing factor in epileptic seizures and subsequent neuronal cell death. Soman is an irreversible inhibitor of cholinesterase, triggering epileptic seizures leading to massive neuronal cell death in brain areas, such as the hippocampus and cerebral cortex. Mitochondrial respiratory chain enzymatic assays were performed in hippocampus and cerebral cortex homogenates from mouse brains collected 3 hours, 24 hours, 3 days, and 7 days after soman poisoning. Our results suggest that mitochondrial enzymatic alterations stem more likely from secondary effects of the poisoning, rather than from any fallout effect from neuronal cell death.

Separate and combined effects of hypobaric hypoxia and hindlimb suspension on skeletal homeostasis and hematopoiesis in mice.

PURPOSE: Bone marrow response to an organismal stress is made by orchestrating the interplay between hematopoietic stem/progenitor cells (HSPCs) and mesenchymal stromal cells (MSCs). Neither the cellular nor the molecular factors that regulate this process are fully understood, especially since this mechanism probably varies depending on the type of stress. Herein, we explored the differentiation and fate of MSCs and HSPCs in mice challenged with a hematopoietic stress or a mechanical stress applied separately or in combination. METHODS: Mice were subjected to 4 days of hypobaric hypoxia (hematopoietic challenge) and/or 7 days of hindlimb suspension (stromal challenge) and then sacrificed for blood and bone collection. Using hematological measurements, colony-forming unit assays, bone histomorphometry and array-based multiplex ELISA analysis, we evaluated challenge influences on both MSC and HSPC mobilization, differentiation (osteoblasts, osteoclasts, and mature blood cells) and fate. RESULTS: We found that hypoxia leads to HSPC mobilization and that an imbalance between bone formation and bone resorption accounts for this mobilization. Whilst suspension is also associated with an imbalance between bone formation and bone resorption, it does not induce HSPC mobilization. Then, we revealed cellular interactions by combining hematopoietic and stromal challenges together in mice. We showed that the hypoxia-driven HSPC mobilization is moderated by suspension. Moreover, when applied in a hypoxic environment, suspension offsets bone imbalance. We identified stroma cell-derived factors MIP-1α, HGF and SDF-1 as potent molecular key players sustaining interactions between hindlimb suspension and hypobaric hypoxia. CONCLUSION: Taken together, our data highlight the benefit of combining different types of stress to better understand the interplay between MSCs and HSPCs.

In vitro and in vivo proves of concept for the use of a chemically cross-linked poly(ester-urethane-urea) scaffold as an easy handling elastomeric biomaterial for bone regeneration.

Bone loss can occur as a result of various pathologies, traumas and injuries and poor bone healing leads to functionally debilitating condition, loss of self-sufficiency and deterioration in life quality. Given the increasing incidence of facial trauma and the emergence of new procedural techniques, advanced scaffolds are currently developed as substitutes for bone tissue engineering. In this study, we investigated the capability of a chemically cross-linked ε-caprolactone-based poly(ester-urethane-urea) (PCLU) scaffold to support bone regeneration. In vitro assays demonstrated that PCLU scaffolds could be colonized by cells through direct cell seeding and cell migration from outside to scaffold inside. Moreover, PCLU scaffolds could provide a suitable environment for stem cells proliferation in a 3D spatial arrangement, and allowed osteogenic differentiation under appropriate induction. In vivo results revealed the osteogenic properties of PCLU scaffolds through a drilled-hole femoral bone defect repair improvement in rats. Using histology and microtomography analysis, we showed that PCLU scaffolds fit well the bone cavity and were eventually entrapped between the newly formed trabeculae. Finally, no sign of inflammation or rejection was noticed. We envision that PCLU scaffolds can provide the clinicians with a substitute having appropriate characteristics for the treatment of bone defects.

Long-term consequences of soman poisoning in mice: part 2. Emotional behavior.

The organophosphorus compound soman produces long-lasting epileptic seizure activity which is associated to brain damage, more particularly in the hippocampus and the amygdala. The companion paper (see part 1 in the same journal issue) describes the neuropathology in the amygdala of soman-poisoned mice. The present paper examines the long-term effects of soman poisoning on emotional reactivity in mice, 30 or 90 days after intoxication using behavioral tasks involving amygdala function. The emotional behavior was estimated in animal tests of unconditioned fear (light/dark boxes, elevated plus-maze) and conditioned fear (auditory and contextual response). In the light/dark boxes and elevated plus-maze, mice intoxicated with soman (110 microg/kg, 1.2 LD(50)) showed an anxiety-like behavior profile at post-poisoning days 30 and 90. In conditioned fear, results showed that both auditory and contextual conditioned responses are increased on post-soman day 30 but no longer on post-soman day 90, evidencing behavioral recovery overtime. This latter behavioral result is in accordance with the delayed neuronal regeneration patterns described in the companion paper (part 1).

Long-term consequences of soman poisoning in mice Part 1. Neuropathology and neuronal regeneration in the amygdala.

To date, studies on soman-induced neuropathology mainly focused on the hippocampus, since this brain region is a well-delimited area with easily detectable pyramidal neurons. Moreover, the hippocampus is severely damaged after soman exposure leading to a substantial alteration of behavioral mnemonic processes. The neuropathology described in the hippocampus, however, and its behavioral consequences cannot be extrapolated to all other limbic damaged brain areas such as the amygdala. Accordingly, in this inaugural paper, using hemalun-phloxin staining and NeuN immunohistochemistry, the number of damaged and residual healthy neurons was quantified in the amygdala in mice over a 90-day period after soman injection (1.2LD(50) of soman). On post-soman day 1, a moderate neuronal cell death (about 23% of the whole neurons) was evidenced. In parallel, a large quantity of degenerating neurons (about 36% of the whole neurons) occurred in this brain region and survived from post-soman day 1 to day 15. The death of these damaged neurons was initiated on post-soman day 30, and ended on post-soman day 90. Concomitantly, as quantified by NeuN immunohistochemistry, a clear neuronal regeneration was demonstrated in the amygdala of soman-poisoned mice between 60 and 90 days after neurotoxicant exposure. In the companion paper (see part 2), the possible effects of both long-term neuropathology and delayed neuronal regeneration were evaluated on amygdala-driven emotional processes.

Soman poisoning induces delayed astrogliotic scar and angiogenesis in damaged mouse brain areas.

Gliotic scar formation and angiogenesis are two biological events involved in the tissue reparative process generally occurring in the brain after mechanically induced injury, ischemia or cerebral tumor development. For the first time, in this study, neo-vascularization and glial scar formation were investigated in the brain of soman-poisoned mice over a 3-month period after nerve agent exposure (1.2 LD50 of soman). Using anti-claudin-5 and anti-vascular endothelial growth factor (VEGF) immunostaining techniques on brain sections, blood vessels were quantified and VEGF expression was verified to appraise the level of neo-angiogenesis induced in damaged brain areas. Furthermore, glial scar formation and neuropathology were estimated over time in the same injured brain regions by anti-glial fibrillary acidic protein (GFAP) immunohistochemistry and hemalun-phloxin (H&P) dye staining, respectively. VEGF over-expression was noticed on post-soman day 3 in lesioned areas such as the hippocampal CA1 field and amygdala. This was followed by an increase in the quantity of mature blood vessels, 3 months after soman poisoning, in the same brain areas. On the other hand, massive astroglial cell activation was demonstrated on post-soman day 8. Reactive astroglial cells were located only in damaged cerebral regions where H&P-stained eosinophilic neurons were found. For longer experimental times, astroglial response slowly decreased overtime but remained detectable on post-soman day 90 in some discrete brain regions (i.e. CA1 field and amygdala) evidencing the formation of a glial scar. In this study, we discuss the key role of VEGF in the angiogenic process and in the glial or neuronal response induced by soman poisoning.

Long-term behavioral consequences of soman poisoning in mice.

We investigated the long-term (up to 90 days) consequences of soman intoxication in mice on weight, motor performances (grip strength, rotarod) and mnemonic cognitive processes (T-maze, Morris water maze test). First, a relative weight loss of 20%, measured 3 days after intoxication, was evidenced as a threshold beyond which neuropathological damage was observed in the hippocampus. Animals were then distributed into either low weight loss (LWL) or high weight loss (HWL) groups according to the relative 20% weight loss threshold. Compared to controls, both groups of poisoned mice quickly exhibited a decrease in their motor performance subsequent to an acute soman toxicity phase. Then, total motor recovery occurred for the LWL group. Comparatively, HWL mice showed only transient recovery prior to a second decrease phase due to soman-induced delayed toxicity. One month after intoxication, mnemonic cognitive performances of the LWL group were similar to controls while the HWL group did not exhibit any learning skill. Three months after poisoning, compared to controls, the LWL group showed similar mnemonic performances in the maze test but a mild deficit in the Morris water maze task. At the same time, learning skills slightly recovered in the HWL group. Mnemonic cognitive data are discussed in relation to the neuropathology, neurogenesis and sprouting occurring in the hippocampus of soman-intoxicated animals.

Early reduction of NeuN antigenicity induced by soman poisoning in mice can be used to predict delayed neuronal degeneration in the hippocampus.

The neuronal nuclei (NeuN) antigen is increasingly being used as a specific marker to identify neuronal cell loss under various pathological conditions. However, recent studies pointed out that a decrease in NeuN labeling could also be due to the reduction of protein expression level or loss of antigenicity and this was not necessarily related to neuronal cell disappearance. We also investigated the presence of damaged neurons, the loss of NeuN immunoreactivity and the level of NeuN protein in the brain hippocampus of mice subjected to soman poisoning (1.2 LD50 of soman). Damaged neurons were detected using hemalun-phloxin (H&P) and Fluoro-Jade B (FJB) staining on brain sections. NeuN immunohistochemistry was also performed on adjacent brain sections and NeuN protein level quantified by Western blot analysis. One and eight days after soman exposure, about 49% of hippocampal neurons were damaged, as assessed by H&P or FJB staining. NeuN immunohistochemistry indicated that all these damaged neurons were deprived of NeuN immunoreactivity. Using Western blot analysis, we proved that loss of NeuN immunoreactivity in degenerating neurons was due to reduced NeuN antigenicity rather than a fall in protein expression level. In this study, we discuss the potential use of NeuN immunohistochemistry as a good biomarker to predict delayed neuronal degeneration in the rodent hippocampus after various brain injuries.