Gabriella Muwanga

All Publications

• Acoustomechanically activatable liposomes for ultrasonic drug uncaging. bioRxiv : the preprint server for biology Purohit, M. P., Roy, K. S., Xiang, Y., Yu, B. J., Azadian, M. M., Muwanga, G., Hart, A. R., Taoube, A. K., Lopez, D. G., Airan, R. D. 2023

  Abstract

  Ultrasound-activatable drug-loaded nanocarriers enable noninvasive and spatiotemporally-precise on-demand drug delivery throughout the body. However, most systems for ultrasonic drug uncaging utilize cavitation or heating as the drug release mechanism and often incorporate relatively exotic excipients into the formulation that together limit the drug-loading potential, stability, and clinical translatability and applicability of these systems. Here we describe an alternate strategy for the design of such systems in which the acoustic impedance and osmolarity of the internal liquid phase of a drug-loaded particle is tuned to maximize ultrasound-induced drug release. No gas phase, cavitation, or medium heating is necessary for the drug release mechanism. Instead, a non-cavitation-based mechanical response to ultrasound mediates the drug release. Importantly, this strategy can be implemented with relatively common pharmaceutical excipients, as we demonstrate here by implementing this mechanism with the inclusion of a few percent sucrose into the internal buffer of a liposome. Further, the ultrasound protocols sufficient for in vivo drug uncaging with this system are achievable with current clinical therapeutic ultrasound systems and with intensities that are within FDA and society guidelines for safe transcranial ultrasound application. Finally, this current implementation of this mechanism should be versatile and effective for the loading and uncaging of any therapeutic that may be loaded into a liposome, as we demonstrate for four different drugs in vitro, and two in vivo. These acoustomechanically activatable liposomes formulated with common pharmaceutical excipients promise a system with high clinical translational potential for ultrasonic drug uncaging of myriad drugs of clinical interest.

  View details for DOI 10.1101/2023.10.23.563690

  View details for PubMedID 37961368

  View details for PubMedCentralID PMC10634775

• Sex-distinct microglial activation and myeloid cell infiltration in the spinal cord after painful peripheral injury. Neurobiology of pain (Cambridge, Mass.) Huck, N. A., Donovan, L. J., Shen, H., Jordan, C. E., Muwanga, G. P., Bridges, C. M., Forman, T. E., Cordonnier, S. A., Haight, E. S., Dale-Huang, F., Takemura, Y., Tawfik, V. L. 2022; 12: 100106

  Abstract

  Chronic pain is a common and often debilitating problem that affects 100 million Americans. A better understanding of pain's molecular mechanisms is necessary for developing safe and effective therapeutics. Microglial activation has been implicated as a mediator of chronic pain in numerous preclinical studies; unfortunately, translational efforts using known glial modulators have largely failed, perhaps at least in part due to poor specificity of the compounds pursued, or an incomplete understanding of microglial reactivity. In order to achieve a more granular understanding of the role of microglia in chronic pain as a means of optimizing translational efforts, we utilized a clinically-informed mouse model of complex regional pain syndrome (CRPS), and monitored microglial activation throughout pain progression. We discovered that while both males and females exhibit spinal cord microglial activation as evidenced by increases in Iba1, activation is attenuated and delayed in females. We further evaluated the expression of the newly identified microglia-specific marker, TMEM119, and identified two distinct populations in the spinal cord parenchyma after peripheral injury: TMEM119+microglia and TMEM119- infiltrating myeloid lineage cells, which are comprised of Ly6G+neutrophils and Ly6G- macrophages/monocytes. Neurons are sensitized by inflammatory mediators released in the CNS after injury; however, the cellular source of these cytokines remains somewhat unclear. Using multiplex in situ hybridization in combination with immunohistochemistry, we demonstrate that spinal cord TMEM119+microglia are the cellular source of cytokines IL6 and IL1beta after peripheral injury. Taken together, these data have important implications for translational studies: 1) microglia remain a viable analgesic target for males and females, so long as duration after injury is considered; 2) the analgesic properties of microglial modulators are likely at least in part related to their suppression of microglial-released cytokines, and 3) a limited number of neutrophils and macrophages/monocytes infiltrate the spinal cord after peripheral injury but have unknown impact on pain persistence or resolution. Further studies to uncover glial-targeted therapeutic interventions will need to consider sex, timing after injury, and the exact target population of interest to have the specificity necessary for translation.

  View details for DOI 10.1016/j.ynpai.2022.100106

  View details for PubMedID 36531615

• The Tibial Fracture-Pin Model: A Clinically Relevant Mouse Model of Orthopedic Injury. Journal of visualized experiments : JoVE Muwanga, G. P., Siliezar-Doyle, J., Ortiz, A. A., Kaslow, J., Haight, E. S., Tawfik, V. L. 2022

  Abstract

  The tibial fracture-pin model is a mouse model of orthopedic trauma and surgery that recapitulates the complex muscle, bone, nerve, and connective tissue damage that manifests with this type of injury in humans. This model was developed because previous models of orthopedic trauma did not include simultaneous injury to multiple tissue types (bone, muscle, nerves) and were not truly representative of human complex orthopedic trauma. The authors therefore modified previous models of orthopedic trauma and developed the tibial fracture-pin model. This modified fracture model consists of a unilateral open tibial fracture with intramedullary nail (IMN) internal fixation and simultaneous tibialis anterior (TA) muscle injury, resulting in mechanical allodynia that lasts up to 5 weeks post injury. This series of protocols outlines the detailed steps to perform the clinically relevant orthopedic trauma tibial fracture-pin model, followed by a modified hot plate assay to examine nociceptive changes after orthopedic injury. Taken together, these detailed, reproducible protocols will allow pain researchers to expand their toolkit for studying orthopedic trauma-induced pain.

  View details for DOI 10.3791/63590

  View details for PubMedID 35969043

• Mechanical Conflict-Avoidance Assay to Measure Pain Behavior in Mice. Journal of visualized experiments : JoVE Gaffney, C. M., Muwanga, G., Shen, H., Tawfik, V. L., Shepherd, A. J. 2022

  Abstract

  Pain comprises of both sensory (nociceptive) and affective (unpleasant) dimensions. In preclinical models, pain has traditionally been assessed using reflexive tests that allow inferences regarding pain's nociceptive component but provide little information about the affective or motivational component of pain. Developing tests that capture these components of pain are therefore translationally important. Hence, researchers need to use non-reflexive behavioral assays to study pain perception at that level. Mechanical conflict-avoidance (MCA) is an established voluntary non-reflexive behavior assay, for studying motivational responses to a noxious mechanical stimulus in a 3 chamber paradigm. A change in a mouse's location preference, when faced with competing noxious stimuli, is used to infer the perceived unpleasantness of bright light versus tactile stimulation of the paws. This protocol outlines a modified version of the MCA assay which pain researchers can use to understand affective-motivational responses in a variety of mouse pain models. Though not specifically described here, our example MCA data use the intraplantar complete Freund's adjuvant (CFA), spared nerve injury (SNI), and a fracture/casting model as pain models to illustrate the MCA procedure.

  View details for DOI 10.3791/63454

  View details for PubMedID 35253785

• Combined single-molecule fluorescence in situ hybridization and immunohistochemistry analysis in intact murine dorsal root ganglia and sciatic nerve. STAR protocols Li, X., Eadara, S., Jeon, S., Liu, Y., Muwanga, G., Qu, L., Caterina, M. J., Meffert, M. K. 2021; 2 (2): 100555

  Abstract

  Single-molecule fluorescence in situ hybridization (smFISH) allows spatial mapping of gene expression. This protocol presents advances in smFISH fidelity and flexibility in intact murine sensory nervous system tissue. An approach using RNAscope probes allows multiplexing, enhanced target specificity, and immunohistochemistry compatibility. Computational strategies increase quantification accuracy of mRNA puncta with a point spread function for clustered transcripts in the dorsal root ganglion and 3D masking for intermingled sciatic nerve cell types. Approaches are validated for mRNAs of modest (Lin28a) and medium (Ppib) steady-state abundance in neurons.

  View details for DOI 10.1016/j.xpro.2021.100555

  View details for PubMedID 34142098

  View details for PubMedCentralID PMC8185307