United States

Background
Radiation therapy significantly impacts the success and cosmetic outcome of implant-based breast reconstruction by inducing irreversible alterations in the surrounding tissues. These changes can include fibrosis, radiation dermatitis, tissue damage, and fat necrosis, which collectively impair the reconstructive process. A common complication is the formation of a dense, fibrotic capsule around the implant, known as symptomatic capsular contracture, which can result in breast firmness, asymmetry, and suboptimal aesthetic outcomes. Radiated tissue also demonstrates reduced elasticity, impaired vascularity, and higher susceptibility to wound healing complications such as dehiscence, infection, and implant loss. Reoperation on irradiated pockets is particularly high-risk due to diminished regenerative capacity, increased fibrosis, and poor tissue quality.
Case Presentation
A patient presented with significant post-radiation asymmetry following bilateral implant-based breast reconstruction. The left, radiated breast developed a tight, contracted implant pocket, resulting in a smaller, higher-riding, and firmer breast mound than the contralateral side. Attempts to revise the radiated side were contraindicated due to concerns about implant exposure, infection, and further tissue compromise. To address the asymmetry, reconstructive efforts focused on modifying the non-radiated breast. The original 500 cc implant was replaced with a 480 cc ultra high-profile Mentor MemoryGel implant, which was tightly wrapped in two large perforated acellular dermal matrix (ADM) sheets. These sheets were configured in a bouffant-style to mimic the fibrotic, projected contour of the radiated side. The ADM was anchored high on the chest wall using 2-0 PDS sutures for secure positioning. Additional skin was excised, and the implant capsule was reinforced to prevent bottoming out, ultimately achieving a symmetric result. 
Discussion
This case presentation illustrates the reconstructive challenges posed by radiation therapy, particularly the inability to safely operate on radiated tissue due to compromised vascularity and structural integrity. Instead of correcting the radiated side directly, the surgical team mirrored its shape and projection by modifying the non-radiated breast. The use of ADM provided structural support and allowed precise shaping to achieve symmetry. This strategic approach addressed both aesthetic and functional concerns while minimizing risk. For patients with similar post-radiation asymmetries, individualized planning that avoids direct manipulation of radiated tissue and instead focuses on contralateral adjustments can provide more optimal outcomes.


AMA Research Challenge 2025 

Achieving Breast Symmetry in Implant Based Reconstruction following Radiation by Manipulating the Non-irradiated Breast


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Background: Glioblastoma multiforme (GBM) is an aggressive grade IV glioma with poor prognosis.1 The PI3K/AKT/mTOR signaling pathway is frequently dysregulated in GBM, contributing to tumor growth, stem cell survival, and immune evasion.2,3,4 This review examines the potential of this pathway to induce apoptosis and improve therapeutic outcomes.

Methods: A literature search was conducted using PubMed to identify articles published between 2018 and 2025. Search terms included “glioblastoma,” “PI3K/AKT/mTOR,” “PI3K therapy,” and “GBM tumor progression.”

Results: Mechanistic studies identify SLC25A32, β2-microglobulin (B2M), and PDIA4 as key regulators of the PI3K pathway in glioblastoma stem cell (GSC) survival. In GSCs, PI3Kα inhibitor, Alpelisib, and mTOR inhibitor, OSI-027, reduced proliferation, underscoring the pathway’s importance.4 SLC25A32, a mitochondrial folate transporter, was elevated in glioma tissue and associated with increased AKT/mTOR phosphorylation.5 B2M, upregulated in GSCs, activated the pathway by increasing PIP2 levels and promoted M2-like tumor-associated macrophage polarization, contributing to immune suppression.6 PDIA4, an ER chaperone, enhanced glycolysis and suppressed pro-apoptotic proteins, supporting GBM progression.7 A Phase I trial of Perifosine (AKT inhibitor) and Temsirolimus (mTOR inhibitor) showed synergistic tumor reduction in GBM patients, though lack of pharmacokinetic data limits mechanistic insights.8

Conclusion: Targeting the PI3K/AKT/mTOR pathway through regulators like SLC25A32, B2M, and PDIA4 shows promise in disrupting GBM survival mechanisms. Dual inhibition strategies may enhance treatment efficacy, but further mechanistic and clinical studies are needed to optimize therapeutic approaches.


Works Cited:
Wu W, Klockow JL, Zhang M, et al. Glioblastoma multiforme (GBM): An overview of current therapies and mechanisms of resistance. Pharmacol Res. 2021;171:105780. doi:10.1016/j.phrs.2021.105780
Berro A, Assi A, Farhat M, et al. Unlocking hope: Anti-VEGFR inhibitors and their potential in glioblastoma treatment. Crit Rev Oncol Hematol. 2024;198:104365. doi:10.1016/j.critrevonc.2024.104365
Agnihotri TG, Salave S, Shinde T, Srikanth I, Gyanani V, Haley JC, Jain A. Understanding the role of endothelial cells in brain tumor formation and metastasis: a proposition to be explored for better therapy. J Natl Cancer Cent. 2023;3(3):222-235. doi:10.1016/j.jncc.2023.08.001.
Eckerdt FD, Bell JB, Gonzalez C, et al. Combined PI3Kα-mTOR Targeting of Glioma Stem Cells. Sci Rep. 2020;10(1):21873. Published 2020 Dec 14. doi:10.1038/s41598-020-78788-z
Xue Z, Wang J, Wang Z, et al. SLC25A32 promotes malignant progression of glioblastoma by activating PI3K-AKT signaling pathway. BMC Cancer. 2023;23(1):589. Published 2023 Jun 26. doi:10.1186/s12885-023-11097-6
Li D, Zhang Q, Li L, et al. β2-Microglobulin Maintains Glioblastoma Stem Cells and Induces M2-like Polarization of Tumor-Associated Macrophages. Cancer Res. 2022;82(18):3321-3334. doi:10.1158/0008-5472.CAN-22-0507
Wang M, Zhang W, Liu Y, et al. PDIA4 promotes glioblastoma progression via the PI3K/AKT/m-TOR pathway. Biochem Biophys Res Commun. 2022;597:83-90. doi:10.1016/j.bbrc.2022.01.115
Kaley TJ, Panageas KS, Pentsova EI, et al. Phase I clinical trial of temsirolimus and perifosine for recurrent glioblastoma. Ann Clin Transl Neurol. 2020;7(4):429-436. doi:10.1002/acn3.51009


Targeting the PI3K/mTOR/AKT Pathway to Induce Apoptosis in Glioblastoma Multiforme: A Mechanistic Review

Over 65 million people live with epilepsy. The most devastating consequence of epilepsy is sudden death. The cause of sudden unexpected death in epilepsy (SUDEP) is unknown. Convulsive seizures can impair awareness and induce respiratory failure. SUDEP usually occurs at night with victims found prone in bed. Brain 5-HT neurons regulate seizure severity, respiratory physiology, are required for awakening to rising CO2 (CO2 arousal). In humans and nocturnal rodents, 5-HT levels are lowest at night. Mouse models of induced and spontaneous seizures (including the Scn1aR1407X/+ model of Dravet Syndrome (DS mice)) are more fatal at night. We hypothesized that the timing of seizure-associated death is regulated by a circadian rhythm and time-of-day variability in 5-HT. To address this, we employed three seizure models (DS mice, MES, and amygdala kindling) and two methods to eliminate brain 5-HT (genetic and chemical elimination). DS mice were used for spontaneous seizure experiments, and Lmx1bf/f/p (KO) or wild-type (WT) littermates were used for induced seizure experiments. KO mice lack >99% of brain 5-HT neurons. DS mice were continuously monitored in constant darkness. Spontaneous sudden deaths exhibited circadian timing, which was greatest during the subjective dark-phase. Deaths occurred more often while mice were awake, and the timing of death was independent of seizure duration, severity, and the timing of potentially fatal seizures. KO mice and WT littermates were subjected to one MES seizure at different times of day. WT mice were more likely to die during the night. However, KO mice had high mortality regardless of time of day. A cohort of DS mice received daily injections of the tryptophan hydroxylase inhibitor para-chlorophenylalanine (800 mg/kg, PCPA) or volume-matched saline. Saline-treated DS mice were more likely to die during the dark-phase, compared to PCPA-treated mice, which died more during the light-phase. Potentially fatal seizures were associated with full hindlimb extension and prolonged recovery. Recovery took longest when seizures occurred during the subjective nighttime. WT mice instrumented for amygdala kindling were used to examine the role of time-of-day seizures on CO2 arousal. Daytime seizures in kindled mice impaired CO2 arousal; however, nighttime seizures profoundly impaired CO2 arousal (over 10-fold increase). SUDEP occurs more frequently during the night and victims are often found in the prone position. Nighttime seizures may exacerbate 5-HT-associated arousal physiology and increase risk of death. Identification of mechanism of death and biomarkers that predict may help develop chronotherapeutic strategies to eliminate nighttime risk of SUDEP.

The nighttime risk of seizure-associated death is influenced by serotonin and coincident with ...

Background
Despite current treatment strategies for atherosclerosis focusing on lifestyle modification and cholesterol reduction, extracoronary vascular diseases affects >200 million patients worldwide and often remain indolent before progressing to severe disease with morbid complications. The aims of this cohort study were to (1) characterize biomarkers associated with development of severe outcomes and (2) compare pathways driving disease progression in different vascular beds in individuals with existing extracoronary vascular disease. 
Methods
We used proteomic data from individuals in the UK Biobank with extracoronary vascular disease at baseline, defined as peripheral artery disease (PAD), carotid artery stenosis (CAS), and renal artery stenosis (RAS). Severe outcomes were defined by major adverse limb events (MALE; developed after PAD), stroke (after CAS), and hemodialysis (after RAS). The first aim utilized Cox proportional hazards regression models to estimate the associations of 1,463 proteins and 249 metabolites with adjustment for age at enrollment, age at prevalent disease diagnosis, sex, cigarette smoking, systolic blood pressure, and statin prescriptions. The second aim utilized Gene Ontology and hypergeometric enrichment tools to identify enriched pathways. 
Results
There were 1370 individuals with a primary diagnosis of extracoronary vascular disease and 1463 individuals with a secondary complication. Patient with secondary complications were more likely to be older (primary diagnosis mean age = 75.63, secondary complication mean age = 76.36) with similar distributions in sex (P = 0.18), body mass index (P = 0.15), and systolic blood pressure (P = 0.40). There were 17 proteins significantly associated (P<0.001) with MALE and 15 proteins significantly associated with stroke (P<0.001). Progression of PAD and MALE was associated with proteins underlying biomineral tissue development, endothelial cell processes, and pathways involved extracellular signal-regulated kinases 1 and 2 (ERK1/2), mitogen-activated protein kinase (MAPK), interleukin-17 (IL-17), phosphoinositide 3-kinase/protein kinase B (PI3K/PKB), and signal transducer and activator of transcription (STAT). Progression of any severe complications, encompassing MALE, stroke, and hemodialysis were associated with proteins involved in vasculogenesis, endothelial cell migration, connective tissue wound healing, collagen catabolism, and pathways involving tumor necrosis factor (TNF) and nuclear factor kappa-light-chain-enhancer of activated B cells (NK-kB). 
Conclusions
Overall, severe complications of extracoronary vascular disease were associated with proteins involved in angiogenesis (PI3K/PKB, STAT), inflammation (IL-17, TNF, NK-kB), and remodeling (biomineral tissue development, connective tissue wound healing, collagen catabolism). This study provides evidence of the complex role of biomarkers in extracoronary vascular disease pathogenesis and highlights specific proteins as potential targets to improve therapeutic strategies.

Proteomics associated with development of severe complications in patients with extracoronary vascular diseases

A Rare Case of Iatrogenic Hemothorax Following Thoracentesis Secondary to an Aberrant Posterior Intercostal Artery


Background

Thoracentesis is the initial procedure performed when diagnosing and treating a pleural effusion. The most common complication for this procedure is pneumothorax, with an incidence reported up to 6%, whereas incidence of hemothorax is approximately 1%.


Case Presentation

A 79-year-old female with history of atrial fibrillation, chronic obstructive pulmonary disease, deep vein thrombosis, type 2 diabetes, and early onset myelodysplastic syndrome presented to the emergency department experiencing chest pain and shortness of breath and was admitted to the hospital for atrial fibrillation with rapid ventricular response. Treatment was initiated, including apixaban. Workup revealed a left pleural effusion of unknown origin, so ultrasound-guided diagnostic thoracentesis was successfully performed using a posterolateral approach, and furosemide was initiated.

Within 2 hours, the patient developed extreme dyspnea, becoming hemodynamically unstable, warranting a transfer to intensive care. Repeat chest X-ray revealed a large pleural effusion, so a chest tube was emergently inserted, removing 1 liter of sanguinous fluid.

Given the rapid development of the hemothorax along with the patient’s anticoagulation treatment and recent thoracentesis, a chest computerized tomography angiogram (CTA) was ordered to detect the source of arterial extravasation. It revealed a ruptured aberrant branch of the left 9th intercostal artery coursing inferiorly across the intercostal space and above the 10th rib, which was the cause of the patient’s rapidly developing hemothorax. This artery was treated with coil embolization, which achieved hemostasis and prevented hemothorax recurrence.


Discussion

Hemothorax is a rare complication of thoracentesis when standard technique based on foundational anatomical knowledge is followed. Proper technique involves inserting the needle above the superior rib margin to avoid the neurovascular bundle. Notably, therapeutic anticoagulation is not a contraindication to thoracentesis and has not been shown to increase bleeding risk. The use of imaging guidance reduces the risk of procedural complication; however, anatomical variants and age-related vascular changes, such as increased tortuosity of intercostal vessels, increase the risk. Vessel tortuosity makes medial approaches more hazardous, supporting the recommendation for a posterolateral entry point in the elderly population. 

In the event of suspected vascular injury, as seen in this case, CTA is the diagnostic modality of choice. When active extravasation is identified, coil embolization serves as the preferred intervention to achieve hemostasis and prevent recurrence. This case illustrates a rare and serious complication of thoracentesis secondary to a previously undocumented anatomical variant of the posterior intercostal artery leading to a nearly fatal hemothorax despite appropriate thoracentesis technique.


Safe Technique, Hidden Danger: Post-Thoracentesis Hemothorax from an Intercostal Variant

Background:
The aftermath of whiplash injuries from motor vehicle accidents (MVA) is estimated to cost nearly $4 billion annually in the United States and inflates over sevenfold with litigation costs. In the relationship between whiplash and litigation, prior research cites that no-fault systems generally limit the opportunity for further litigation. However, New York State utilizes a no-fault system that may award plaintiffs further compensation based on a serious injury threshold, for which both injury and treatment are considered. Most MVA do not biomechanically cause disc herniations and are conservatively treated, yet these injuries are frequently litigated. This study examines how treatment of cervical disc herniations claimed in no-fault litigation may impact award compensation, thus evaluating overtreatment as a potential tool to increase compensation.

Methods:
We queried the Thomson Reuters Westlaw database for Jury Verdicts & Settlements in New York that were categorized under “no-fault” and “motor vehicle” and included cervical disc herniation in the claim. 181 of 383 cases from 2005-2011 met the injury criteria and had detailed therapeutic methodology, litigation outcome, and award amount. We categorized cervical disc herniation treatments as surgical or non-surgical. We then separated cases that favored the plaintiff to any degree or were resolved alternatively (Plaintiff Verdict, Settlement, Mediated Settlement, Arbitration, Mixed Verdict) from cases with defendant-oriented outcomes (Defendant Decision and Defendant Verdict). The mean and median true awards (actual value collected by the plaintiff, potentially adjusted by pre-trial high-low agreement) were compared across groups and the Mann-Whitney U Test was used to test significance (p<0.05).

Results:
Of the 181 cases that met our initial query criteria, we included 18 cases describing surgical treatment and 80 cases describing non-surgical treatment that resulted in a plaintiff-oriented outcome. Analysis of included cases demonstrated a significantly greater true award for surgical treatment(s) (n=18, M=$959,257.19, Mdn=$637,500) compared to non-surgical treatment(s) (n=80, M=$195,184.10, Mdn=$74,250, U=141, p<0.001). There was no significant correlation to suggest that either treatment group more often resulted in a defendant or plaintiff-oriented outcome.

Conclusion:
Substantial financial incentives may exist for cervical disc surgical intervention in whiplash compared to more conservative, non-surgical treatment. While neither treatment group resulted in significantly more plaintiff-oriented outcomes, the compensation potential for invasive intervention may still prompt overtreatment. Further work is needed to determine how the medical and legal communities can minimize the economic toll of whiplash while ensuring patients are receiving appropriate care and just compensation.

Impact of Surgical Treatment on Award in NYS No-Fault Accident Claims of Cervical Disc Herniation

Background
Infertility is especially prevalent among female surgeons at all levels of training. Given the high rates of infertility and delayed childbearing among female surgeons, many must utilize assisted reproductive technology (ART) to help them achieve pregnancy. Insurance plans may help offset the financial burden of ART, but coverage has often been limited and inconsistent. This study aims to assess the current status of insurance coverage for ART in orthopaedic surgery residency programs.

Methods
This is a cross-sectional study of 218 orthopaedic surgery residency programs in the United States in 2024. Program websites were searched for resident benefits and insurance coverage for fertility services and dollar coverage for procedures and prescriptions. Program coordinators were identified, and a survey was distributed in addition to phone inquiries about incomplete information and supplemental funding outside of default insurance coverage.

Results
A total of 118 (54.1%) programs had publicly available or available upon request information on insurance coverage of ART. Of those, 106 (90%) had some form of fertility service coverage. Regarding service eligibility, 44 (41.5%) programs required preauthorization, while 15 (14.2%) programs required referrals. IVF and IUI were the most commonly offered ART services, with 75.5% and 71.7% coverage, respectively. The median number of cycles covered was 4 (Q1: 3, Q3: 4), and the median coverage amount was $21,000 (Q1: $15,000; Q3: $30,000). Spousal coverage was offered by 29 (27.4%) programs. Supplemental funding was available at 6 (5.7%) programs.

Conclusion
This study highlights multiple barriers that orthopaedic surgery trainees face to access ART. In addition to limited information availability, there is variation in coverage for adjunct services and minimal supplemental funding from programs. Additional eligibility barriers exist via pre-authorization or referral requirements. Moreover, the median coverage amount may not provide sufficient coverage for some cases. Programs can improve access to fertility treatments and care by promoting transparency and providing supplemental funding.

Assisted Reproductive Technology (ART) Coverage in Orthopaedic Training Programs

Background: The uninsured population of Galveston is estimated at 18.3% of the total 53,000 individuals. Approximately 400 unhoused individuals dwell on the island, comprising .8% of the population. The Luke Society’s mission is to provide care to these patients. Luke’s provides physician consultations, medication, and comprehensive wound care. This study aims to analyze the wounds treated at this free clinic to guide public health interventions in vulnerable populations of Galveston, TX.
Methods: Twenty-six care encounters were documented between September 2024 and February 2025. Data including wound description, location, treatment, and materials provided were recorded. No demographic data or patient identifiers were collected.
Results: 38% of wounds involved the feet and 19% affected the head. 46% were infected and treated with either antibiotics or antifungals. 27% were related to a specific trauma. 73% of patients received materials for wound care and cleaning. 4 of the 26 were post surgical wounds that had been lost to follow up by their surgeon. 
Conclusions: The high prevalence of foot related wounds indicates a lack of sufficient hygiene likely attributed to the high rates of homelessness and a lack of access to consistent showers and clean socks. Head injuries could potentially be prevented with better access to helmets as many of the patients travel on bikes. Underinsured patients have a high rate of being lost to follow up leading to complications and infections following surgeries.
Public Health Implications: Preventative medicine and regular cleaning is highly important in wound care and trauma related injuries. Access to clean socks, helmets, warm water, and basic wound care supplies should be a focus in community health initiatives supporting the underinsured and unhoused populations. With these interventions, chronic wounds could heal faster and potential progression to serious infections.


Wound Care Trends of a Student Run Clinic in the Underinsured and Unhoused Population of Galveston, Texas

Background
The American Diabetes Association recommends annual diabetic retinopathy screening to prevent vision loss, yet national screening rates remain below the Healthy People 2030 target of 70%. Screening rates for diabetic retinopathy at Northwestern Medicine averaged 49.7% in June 2023, indicating care gaps across Chicagoland. 

Methods
Our quality improvement initiative utilized Plan-Do-Study-Act cycles, root cause analysis, and data-driven decision-making. Interventions were applied to both regional Northwestern Medicine primary care clinics and the primary care clinics of the downtown Northwestern academic center. Key interventions included pre-visit questionnaires, practice advisory tools, more frequent health maintenance standards, patient attestations, and automated MyChart messaging. The effectiveness of each intervention was assessed by aligning implementation timing with monthly screening rates. A team of quality improvement professionals evaluated each initiative's impact versus effort to determine scalability.

Results
Screening rates increased from 49.7% to 57% at the downtown campus and from 55% to 59% at regional clinics over a 22-month period.

At the downtown academic center, the implementation of a pre-visit questionnaire (allows patients to report recently completed or upcoming eye exams) in August 2024 increased rates of screening from 50% to 54% within one month. Implementation of patient attestations (allows for self-reporting of completed eye exam status in MyChart) and automated MyChart messaging (messages and scheduling options proactively sent to patients overdue for eye exam) further increased screening rates to 57% over the next 7 months. 

At the regional Northwestern primary care clinics, a standardized practice advisory (notifies staff that a patient is due for an eye exam and prompts them to set up an ophthalmology referral) introduced in June 2023 through March 2025 demonstrated steadily increasing screening rates from 55% to 59%. Additionally, updating the health maintenance frequency (requiring annual diabetic eye exams for all patients with diabetes) in September 2024 demonstrated an almost 1% increase within one month. 

High-impact, low-effort interventions included the pre-visit questionnaire, updated health maintenance frequencies, MyChart messaging, and patient attestations. Resource-intensive but effective measures also included integrating community health workers and a point-of-care camera at the downtown site. Less impactful strategies included distribution of educational materials and a self-scheduling feature.

Conclusion
This initiative highlights effective, scalable strategies for improving diabetic retinopathy screening rates at both academic and regional clinics. These findings may guide similar efforts at other health care systems to address gaps in preventive diabetes care.


Quality Improvement Methods for Improving Diabetic
Retinopathy Screening Rates at a Large Academic Medical
Center and Affiliate Medical Group

Background: Lymphomatoid papulosis (LyP) is a chronic, recurrent, self-healing cutaneous T-cell lymphoproliferative disorder characterized by CD30+ cells. It typically presents with papulonodular eruptions rather than plaques. Mycosis fungoides (MF), a primary cutaneous T-cell lymphoma, is generally persistent and non-migratory. This case highlights an unusual presentation of migratory plaque-like LyP, raising diagnostic challenges with overlap to more serious lymphoproliferations, such as MF.

Case Presentation: A 63-year-old female presented with pruritic plaques and nodules that intermittently appeared and resolved across various body sites. Past episodes were treated as fungal infections, though KOH preps were negative. She had a remote history of shingles but no other relevant systemic symptoms. Two punch biopsies were obtained a week apart, one from the right thigh and the other from the right groin.

Histopathology from both biopsies revealed an atypical intraepidermal T-cell lymphoproliferative disorder with CD30 positivity and a double-negative CD4-/CD8- phenotype by immunohistochemistry. Loss of several T-cell markers (CD2, CD5, CD7) was noted. The dermal component demonstrated a band-like infiltrate of mixed immunophenotype, including CD30 positive lymphoid cells. PAS was negative for fungal elements. T-cell receptor studies were positive for TCR beta clones (261 and 260bp; 294 and 294bp) as well as TCR gamma clones (240 and 239bp). Ancillary FISH for the DUSP22-IRF4 rearrangement is pending. Given the migratory nature of the rash, self-resolution of lesions, and histopathologic work-up with ancillary studies, a diagnosis of an unusual plaque-like variant of lymphomatoid papulosis was made.

Discussion: This case underscores a diagnostic gray zone between LyP and MF. Plaque-like LyP is rare and may be underrecognized due to its clinical resemblance to early MF. The migratory nature of the lesions and spontaneous resolution pointed toward LyP, yet the presence of plaques and immunophenotypic abnormalities added complexity. CD30+ double-negative T-cell populations are uncommon but can occur in LyP, occasionally associated with DUSP22-IRF4 rearrangements. Accurate diagnosis is crucial given the differing prognoses and management strategies between LyP and MF. This case also illustrates the value of serial biopsies and multidisciplinary pathology review in evaluating atypical cutaneous lymphoproliferative disorders.

Migratory Plaque-Like Lymphomatoid Papulosis: A Rare Clinical Presentation Mimicking Mycosis

Background
Repetitive traumatic brain injury (rTBI) is a leading risk factor for chronic traumatic encephalopathy (CTE) and other neurodegenerative disorders. While CTE has traditionally been associated with repeated head impacts, evidence suggests that a single TBI may drive tau pathology and progressive neurodegeneration. Biological sex is also known to influence TBI outcomes, yet mechanisms underlying sex differences remain poorly defined. Existing animal models often lack reproducibility and fail to include both sexes. This study aimed to develop and validate a rigorously calibrated Drosophila melanogaster model of rTBI to investigate sex-specific behavioral, molecular, and neuropathological responses to repetitive head trauma.

Methods
An automated, CO₂-driven ballistic impactor was engineered to deliver reproducible closed-head injuries to male and female flies. Flies received five impacts per day 1 psi to model mild rTBI. Locomotor activity and cognitive function were assessed using automated tracking and value-based feeding decision paradigms, respectively. Neurodegeneration was evaluated histologically, and whole-head proteomic profiling was performed at multiple time points post-injury to identify rTBI and sexually dimorphic molecular pathways.

Results
rTBI produced dose-dependent locomotor deficits and impaired decision-making in both sexes. Females were initially more resilient to multiple injuries, exhibiting fewer locomotor deficits early post-injury, but demonstrated significantly longer recovery periods compared to males. Histological analyses revealed increased neurodegeneration present despite tau knockout (dTauKO) flies, suggesting tau-independent mechanisms. Proteomic analysis revealed sex-specific alterations in mitochondrial function, oxidative stress responses, and pathways regulating locomotion and energy metabolism, which correlated with observed behavioral phenotypes.

Conclusion
This enhanced Drosophila rTBI model provides a robust model for studying sex-specific mechanisms of neurodegeneration. Our findings suggest that both the number of injuries and cumulative force contribute to neuropathology and highlight critical differences in male and female responses to repetitive head trauma. These insights emphasize the importance of incorporating sex as a biological variable in TBI research and identify potential therapeutic targets for mitigating long-term neurological sequelae.

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