Browsing by Author "Haulcomb, Melissa M."
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Item Axotomy-induced target disconnection promotes an additional death mechanism involved in motoneuron degeneration in amyotrophic lateral sclerosis transgenic mice(Wiley, 2014-07) Haulcomb, Melissa M.; Mesnard, Nichole A.; Batka, Richard J.; Alexander, Thomas D.; Sanders, Virginia M.; Jones, Kathryn J.; Department of Anatomy & Cell Biology, School of MedicineThe target disconnection theory of amyotrophic lateral sclerosis (ALS) pathogenesis suggests that disease onset is initiated by a peripheral pathological event resulting in neuromuscular junction loss and motoneuron (MN) degeneration. Presymptomatic mSOD1(G93A) mouse facial MN (FMN) are more susceptible to axotomy-induced cell death than wild-type (WT) FMN, which suggests additional CNS pathology. We have previously determined that the mSOD1 molecular response to facial nerve axotomy is phenotypically regenerative and indistinguishable from WT, whereas the surrounding microenvironment shows significant dysregulation in the mSOD1 facial nucleus. To elucidate the mechanisms underlying the enhanced mSOD1 FMN loss after axotomy, we superimposed the facial nerve axotomy model on presymptomatic mSOD1 mice and investigated gene expression for death receptor pathways after target disconnection by axotomy vs. disease progression. We determined that the TNFR1 death receptor pathway is involved in axotomy-induced FMN death in WT and is partially responsible for the mSOD1 FMN death. In contrast, an inherent mSOD1 CNS pathology resulted in a suppressed glial reaction and an upregulation in the Fas death pathway after target disconnection. We propose that the dysregulated mSOD1 glia fail to provide support the injured MN, leading to Fas-induced FMN death. Finally, we demonstrate that, during disease progression, the mSOD1 facial nucleus displays target disconnection-induced gene expression changes that mirror those induced by axotomy. This validates the use of axotomy as an investigative tool in understanding the role of peripheral target disconnection in the pathogenesis of ALS.Item Evaluation of Low versus High Volume per Minute Displacement CO₂ Methods of Euthanasia in the Induction and Duration of Panic-Associated Behavior and Physiology(MDPI, 2016-08) Hickman, Debra L.; Fitz, Stephanie D.; Bernabe, Cristian S.; Caliman, Izabela F.; Haulcomb, Melissa M.; Federici, Lauren M.; Shekhar, Anatatha; Johnson, Philip L.; Department of Cellular & Integrative Physiology, IU School of MedicineCurrent recommendations for the use of CO ₂ as a euthanasia agent for rats require the use of gradual fill protocols (such as 10% to 30% volume displacement per minute) in order to render the animal insensible prior to exposure to levels of CO ₂ that are associated with pain. However, exposing rats to CO ₂ , concentrations as low as 7% CO ₂ are reported to cause distress and 10%-20% CO ₂ induces panic-associated behavior and physiology, but loss of consciousness does not occur until CO ₂ concentrations are at least 40%. This suggests that the use of the currently recommended low flow volume per minute displacement rates create a situation where rats are exposed to concentrations of CO ₂ that induce anxiety, panic, and distress for prolonged periods of time. This study first characterized the response of male rats exposed to normoxic 20% CO ₂ for a prolonged period of time as compared to room air controls. It demonstrated that rats exposed to this experimental condition displayed clinical signs consistent with significantly increased panic-associated behavior and physiology during CO ₂ exposure. When atmospheric air was then again delivered, there was a robust increase in respiration rate that coincided with rats moving to the air intake. The rats exposed to CO ₂ also displayed behaviors consistent with increased anxiety in the behavioral testing that followed the exposure. Next, this study assessed the behavioral and physiologic responses of rats that were euthanized with 100% CO ₂ infused at 10%, 30%, or 100% volume per minute displacement rates. Analysis of the concentrations of CO ₂ and oxygen in the euthanasia chamber and the behavioral responses of the rats suggest that the use of the very low flow volume per minute displacement rate (10%) may prolong the duration of panicogenic ranges of ambient CO ₂ , while the use of the higher flow volume per minute displacement rate (100%) increases agitation. Therefore, of the volume displacement per minute rates evaluated, this study suggests that 30% minimizes the potential pain and distress experienced by the animal.Item Facial Nerve Axotomy in Mice: A Model to Study Motoneuron Response to Injury(2015-02) Olmstead, Deborah N.; Mesnard-Hoaglin, Nichole A.; Batka, Richard J.; Haulcomb, Melissa M.; Miller, Whitney M.; Jones, Kathryn J.; Department of Anatomy & Cell Biology, IU School of MedicineThe goal of this surgical protocol is to expose the facial nerve, which innervates the facial musculature, at its exit from the stylomastoid foramen and either cut or crush it to induce peripheral nerve injury. Advantages of this surgery are its simplicity, high reproducibility, and the lack of effect on vital functions or mobility from the subsequent facial paralysis, thus resulting in a relatively mild surgical outcome compared to other nerve injury models. A major advantage of using a cranial nerve injury model is that the motoneurons reside in a relatively homogenous population in the facial motor nucleus in the pons, simplifying the study of the motoneuron cell bodies. Because of the symmetrical nature of facial nerve innervation and the lack of crosstalk between the facial motor nuclei, the operation can be performed unilaterally with the unaxotomized side serving as a paired internal control. A variety of analyses can be performed postoperatively to assess the physiologic response, details of which are beyond the scope of this article. For example, recovery of muscle function can serve as a behavioral marker for reinnervation, or the motoneurons can be quantified to measure cell survival. Additionally, the motoneurons can be accurately captured using laser microdissection for molecular analysis. Because the facial nerve axotomy is minimally invasive and well tolerated, it can be utilized on a wide variety of genetically modified mice. Also, this surgery model can be used to analyze the effectiveness of peripheral nerve injury treatments. Facial nerve injury provides a means for investigating not only motoneurons, but also the responses of the central and peripheral glial microenvironment, immune system, and target musculature. The facial nerve injury model is a widely accepted peripheral nerve injury model that serves as a powerful tool for studying nerve injury and regeneration.Item Fast and slow rates of symptom progression in the transgenic SOD1 murine model of ALS(Office of the Vice Chancellor for Research, 2011-04-08) Haulcomb, Melissa M.; Mesnard, Nichole A.; Sanders, Virginia M.; Jones, Kathryn J.ALS is a disease targeting motoneurons (MN). In the SOD1 mouse model of ALS, an axonal dieback process is initiated during the pre-symptomatic stage where MN axons withdraw from target muscle. We have used facial nerve axotomy, which resembles the axonal die-back response, in pre-symptomatic SOD1 mice to investigate aspects of the disease. Apoptotic and pro-inflammatory gene expression is upregulated in pre-symptomatic SOD1 axotomized facial nuclei in addition to significant SOD1 MN death. Disease progression in symptomatic SOD1 facial nuclei resembles the molecular response initiated by axotomy. MN survival levels in symptomatic SOD1 and axotomized, presymptomatic SOD1 facial nuclei are similar. Therefore, facial nerve axotomy produces a disease onset-like response. The current study used behavioral testing to assess motor function, and revealed two groups of SOD1 mice with differing rates of symptomatic disease progression. The slow progression group had significantly less motor impairments compared to the fast progression group, but no difference in symptom onset was seen. Fast progression group showed higher mRNA levels for genes related to axonal injury. Symptomatic severity in SOD1 mice correlates to the cellular and molecular responses to axonal injury. Therefore, research using treatments to slow disease or extend.Item Identification of a resilient mouse facial motoneuron population following target disconnection by injury or disease(IOS, 2018) Setter, Deborah O.; Haulcomb, Melissa M.; Beahrs, Taylor; Meadows, Rena M.; Schartz, Nicole D.; Custer, Sara K.; Sanders, Virginia M.; Jones, Kathryn J.; Anatomy and Cell Biology, IU School of MedicineBackground: When nerve transection is performed on adult rodents, a substantial population of neurons survives short-term disconnection from target, and the immune system supports this neuronal survival, however long-term survival remains unknown. Understanding the effects of permanent axotomy on cell body survival is important as target disconnection is the first pathological occurrence in fatal motoneuron diseases such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). Objective: The goal of this study was to determine if facial motoneurons (FMN) could survive permanent target disconnection up to 26 weeks post-operation (wpo) after facial nerve axotomy (FNA). In addition, the potentially additive effects of immunodeficiency and motoneuron disease on post-axotomy FMN survival were examined. Methods: This study included three wild type (WT) mouse strains (C57BL/6J, B6SJL, and FVB/NJ) and three experimental models (RAG-2-/-: immunodeficiency; mSOD1: ALS; Smn-/-/SMN2+/+: SMA). All animals received a unilateral FNA, and FMN survival was quantified at early and extended post-operative timepoints. Results: In the C57BL/6J WT group, FMN survival significantly decreased at 10 wpo (55 ± 6%), and then remained stable out to 26 wpo (47 ± 6%). In the RAG-2-/- and mSOD1 groups, FMN death occurred much earlier at 4 wpo, and survival plateaued at approximately 50% at 10 wpo. The SMA model and other WT strains also exhibited approximately 50% FMN survival after FNA. Conclusion: These results indicate that immunodeficiency and motoneuron disease accelerate axotomy-induced neuron death, but do not increase total neuron death in the context of permanent target disconnection. This consistent finding of a target disconnection-resilient motoneuron population is prevalent in other peripheral nerve injury models and in neurodegenerative disease models as well. Characterization of the distinct populations of vulnerable and resilient motoneurons may reveal new therapeutic approaches for injury and disease.Item Identification of B6SJL mSOD1(G93A) mouse subgroups with different disease progression rates(Wiley, 2015-12-15) Haulcomb, Melissa M.; Mesnard-Hoaglin, Nichole A.; Batka, Richard J.; Meadows, Rena M.; Miller, Whitney M.; McMillan, Kathryn P.; Brown, Todd J.; Sanders, Virginia M.; Jones, Kathryn J.; Department of Anatomy & Cell Biology, IU School of MedicineDisease progression rates among patients with amyotrophic lateral sclerosis (ALS) vary greatly. Although the majority of affected individuals survive 3-5 years following diagnosis, some subgroups experience a more rapidly progressing form, surviving less than 1 year, and other subgroups experience slowly progressing forms, surviving nearly 50 years. Genetic heterogeneity and environmental factors pose significant barriers in investigating patient progression rates. Similar to the case for humans, variation in survival within the mSOD1 mouse has been well documented, but different progression rates have not been investigated. The present study identifies two subgroups of B6SJL mSOD1(G93A) mice with different disease progression rates, a fast progression group (FPG) and slow progression group, as evidenced by differences in the rate of motor function decline. In addition, increased disease-associated gene expression within the FPG facial motor nucleus confirmed the presence of a more severe phenotype. We hypothesize that a more severe disease phenotype could be the result of 1) an earlier onset of axonal disconnection with a consistent degeneration rate or 2) a more severe or accelerated degenerative process. We performed a facial nerve transection axotomy in both mSOD1 subgroups prior to disease onset as a method to standardize the axonal disconnection. Instead of leading to comparable gene expression in both subgroups, this standardization did not eliminate the severe phenotype in the FPG facial nucleus, suggesting that the FPG phenotype is the result of a more severe or accelerated degenerative process. We theorize that these mSOD1 subgroups are representative of the rapid and slow disease phenotypes often experienced in ALS.Item Impact of peripheral immune status on central molecular responses to facial nerve axotomy(Elsevier, 2018-02) Setter, Deborah O.; Runge, Elizabeth M.; Schartz, Nicole D.; Kennedy, Felicia M.; Brown, Brandon L.; McMillan, Kathryn P.; Miller, Whitney M.; Shah, Kishan M.; Haulcomb, Melissa M.; Sanders, Virginia M.; Jones, Karthryn J.; Anatomy and Cell Biology, IU School of MedicineWhen facial nerve axotomy (FNA) is performed on immunodeficient recombinase activating gene-2 knockout (RAG-2-/-) mice, there is greater facial motoneuron (FMN) death relative to wild type (WT) mice. Reconstituting RAG-2-/- mice with whole splenocytes rescues FMN survival after FNA, and CD4+ T cells specifically drive immune-mediated neuroprotection. Evidence suggests that immunodysregulation may contribute to motoneuron death in amyotrophic lateral sclerosis (ALS). Immunoreconstitution of RAG-2-/- mice with lymphocytes from the mutant superoxide dismutase (mSOD1) mouse model of ALS revealed that the mSOD1 whole splenocyte environment suppresses mSOD1 CD4+ T cell-mediated neuroprotection after FNA. The objective of the current study was to characterize the effect of CD4+ T cells on the central molecular response to FNA and then identify if mSOD1 whole splenocytes blocked these regulatory pathways. Gene expression profiles of the axotomized facial motor nucleus were assessed from RAG-2-/- mice immunoreconstituted with either CD4+ T cells or whole splenocytes from WT or mSOD1 donors. The findings indicate that immunodeficient mice have suppressed glial activation after axotomy, and cell transfer of WT CD4+ T cells rescues microenvironment responses. Additionally, mSOD1 whole splenocyte recipients exhibit an increased astrocyte activation response to FNA. In RAG-2-/- + mSOD1 whole splenocyte mice, an elevation of motoneuron-specific Fas cell death pathways is also observed. Altogether, these findings suggest that mSOD1 whole splenocytes do not suppress mSOD1 CD4+ T cell regulation of the microenvironment, and instead, mSOD1 whole splenocytes may promote motoneuron death by either promoting a neurotoxic astrocyte phenotype or inducing Fas-mediated cell death pathways. This study demonstrates that peripheral immune status significantly affects central responses to nerve injury. Future studies will elucidate the mechanisms by which mSOD1 whole splenocytes promote cell death and if inhibiting this mechanism can preserve motoneuron survival in injury and disease.Item Locomotor analysis identifies early compensatory changes during disease progression and subgroup classification in a mouse model of amyotrophic lateral sclerosis(Medknow Publications, 2017-10) Haulcomb, Melissa M.; Meadows, Rena M.; Miller, Whitney M.; McMillan, Kathryn P.; Hilsmeyer, MeKenzie J.; Wang, Xuefu; Beaulieu, Wesley T.; Dickinson, Stephanie L.; Brown, Todd J.; Sanders, Virginia M.; Jones, Kathryn J.; Anatomy and Cell Biology, School of MedicineAmyotrophic lateral sclerosis is a motoneuron degenerative disease that is challenging to diagnose and presents with considerable variability in survival. Early identification and enhanced understanding of symptomatic patterns could aid in diagnosis and provide an avenue for monitoring disease progression. Use of the mSOD1G93A mouse model provides control of the confounding environmental factors and genetic heterogeneity seen in amyotrophic lateral sclerosis patients, while investigating underlying disease-induced changes. In the present study, we performed a longitudinal behavioral assessment paradigm and identified an early hindlimb symptom, resembling the common gait abnormality foot drop, along with an accompanying forelimb compensatory mechanism in the mSOD1G93A mouse. Following these initial changes, mSOD1 mice displayed a temporary hindlimb compensatory mechanism resembling an exaggerated steppage gait. As the disease progressed, these compensatory mechanisms were not sufficient to sustain fundamental locomotor parameters and more severe deficits appeared. We next applied these initial findings to investigate the inherent variability in B6SJL mSOD1G93A survival. We identified four behavioral variables that, when combined in a cluster analysis, identified two subpopulations with different disease progression rates: a fast progression group and a slow progression group. This behavioral assessment paradigm, with its analytical approaches, provides a method for monitoring disease progression and detecting mSOD1 subgroups with different disease severities. This affords researchers an opportunity to search for genetic modifiers or other factors that likely enhance or slow disease progression. Such factors are possible therapeutic targets with the potential to slow disease progression and provide insight into the underlying pathology and disease mechanisms.Item The Need for Speed in Rodent Locomotion Analyses.(Wiley, 2014-10) Batka, Richard J.; Brown, Todd J.; Mcmillan, Kathryn P.; Meadows, Rena M.; Jones, Kathryn J.; Haulcomb, Melissa M.; Department of Anatomy & Cell Biology, IU School of MedicineLocomotion analysis is now widely used across many animal species to understand the motor defects in disease, functional recovery following neural injury, and the effectiveness of various treatments. More recently, rodent locomotion analysis has become an increasingly popular method in a diverse range of research. Speed is an inseparable aspect of locomotion that is still not fully understood, and its effects are often not properly incorporated while analyzing data. In this hybrid manuscript, we accomplish three things: (1) review the interaction between speed and locomotion variables in rodent studies, (2) comprehensively analyze the relationship between speed and 162 locomotion variables in a group of 16 wild-type mice using the CatWalk gait analysis system, and (3) develop and test a statistical method in which locomotion variables are analyzed and reported in the context of speed. Notable results include the following: (1) over 90% of variables, reported by CatWalk, were dependent on speed with an average R2 value of 0.624, (2) most variables were related to speed in a nonlinear manner, (3) current methods of controlling for speed are insufficient, and (4) the linear mixed model is an appropriate and effective statistical method for locomotion analyses that is inclusive of speed-dependent relationships. Given the pervasive dependency of locomotion variables on speed, we maintain that valid conclusions from locomotion analyses cannot be made unless they are analyzed and reported within the context of speed.Item SOD1G93A transgenic mouse CD4+ T cells mediate neuroprotection after facial nerve axotomy when removed from a suppressive peripheral microenvironment(Elsevier B.V., 2014-08) Mesnard-Hoaglin, Nichole A.; Xin, Junping; Haulcomb, Melissa M.; Batka, Richard J.; Sanders, Virginia M.; Jones, Kathryn J.; Department of Anatomy & Cell Biology, IU School of MedicineAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease involving motoneuron (MN) axonal withdrawal and cell death. Previously, we established that facial MN (FMN) survival levels in the SOD1G93A transgenic mouse model of ALS are reduced and nerve regeneration is delayed, similar to immunodeficient RAG2-/- mice, after facial nerve axotomy. The objective of this study was to examine the functionality of SOD1G93A splenic microenvironment, focusing on CD4+ T cells, with regard to defects in immune-mediated neuroprotection of injured MN. We utilized the RAG2-/- and SOD1G93A mouse models, along with the facial nerve axotomy paradigm and a variety of cellular adoptive transfers, to assess immune-mediated neuroprotection of FMN survival levels. We determined that adoptively transferred SOD1G93A unfractionated splenocytes into RAG2-/- mice were unable to support FMN survival after axotomy, but that adoptive transfer of isolated SOD1G93A CD4+ T cells could. Although WT unfractionated splenocytes adoptively transferred into SOD1G93A mice were able to maintain FMN survival levels, WT CD4+ T cells alone could not. Importantly, these results suggest that SOD1G93A CD4+ T cells retain neuroprotective functionality when removed from a dysfunctional SOD1G93A peripheral splenic microenvironment. These results also indicate that the SOD1G93A central nervous system microenvironment is able to re-activate CD4+ T cells for immune-mediated neuroprotection when a permissive peripheral microenvironment exists. We hypothesize that dysfunctional SOD1G93A peripheral splenic microenvironment may compromise neuroprotective CD4+ T cell activation and/or differentiation, which, in turn, results in impaired immune-mediated neuroprotection for MN survival after peripheral axotomy in SOD1G93A mice.