Expression of MHC II in DRG neurons attenuates paclitaxel-induced cold hypersensitivity in male and female mice.

Chemotherapy is often a life-saving treatment, but the development of intractable pain caused by chemotherapy-induced peripheral neuropathy (CIPN) is a major dose-limiting toxicity that restricts cancer survival rates. Recent reports demonstrate that paclitaxel (PTX) robustly increases anti-inflammatory CD4+ T cells in the dorsal root ganglion (DRG), and that T cells and anti-inflammatory cytokines are protective against CIPN. However, the mechanism by which CD4+ T cells are activated, and the extent cytokines released by CD4+ T cells target DRG neurons are unknown. Here, we are the first to detect major histocompatibility complex II (MHCII) protein in mouse DRG neurons and to find CD4+ T cells breaching the satellite glial cell barrier to be in close proximity to neurons, together suggesting CD4+ T cell activation and targeted cytokine release. MHCII protein is primarily expressed in small nociceptive neurons in male and female mouse DRG but increased after PTX in small nociceptive neurons in only female DRG. Reducing one copy of MHCII in small nociceptive neurons decreased anti-inflammatory IL-10 and IL-4 producing CD4+ T cells in naïve male DRG and increased their hypersensitivity to cold. Administration of PTX to male and female mice that lacked one copy of MHCII in nociceptive neurons decreased anti-inflammatory CD4+ T cells in the DRG and increased the severity of PTX-induced cold hypersensitivity. Collectively, our results demonstrate expression of MHCII protein in mouse DRG neurons, which modulates cytokine producing CD4+ T cells in the DRG and attenuates cold hypersensitivity during homeostasis and after PTX treatment.

Novel expression of major histocompatibility complex II in dorsal root ganglion neurons attenuates paclitaxel-induced cold hypersensitivity in male and female mice.

Chemotherapy is often a life-saving treatment, but the development of intractable pain caused by chemotherapy-induced peripheral neuropathy (CIPN) is a major dose-limiting toxicity that restricts survival rates. Recent reports demonstrate that paclitaxel (PTX) robustly increases anti-inflammatory CD4+ T cells in the dorsal root ganglion (DRG), and that T cells and anti-inflammatory cytokines are protective against CIPN. However, the mechanism by which CD4+ T cells are activated, and the extent cytokines released by CD4+ T cells target DRG neurons are unknown. Here, we found novel expression of functional major histocompatibility complex II (MHCII) protein in DRG neurons, and CD4+ T cells in close proximity to DRG neurons, together suggesting CD4+ T cell activation and targeted cytokine release. MHCII protein is primarily expressed in small nociceptive neurons in male mouse DRG regardless of PTX, while MHCII is induced in small nociceptive neurons in female DRG after PTX. Accordingly, reducing MHCII in small nociceptive neurons increased hypersensitivity to cold only in naive male mice, but increased severity of PTX-induced cold hypersensitivity in both sexes. Collectively, our results demonstrate expression of MHCII on DRG neurons and a functional role during homeostasis and inflammation.

Ovariectomy increases paclitaxel-induced mechanical hypersensitivity and reduces anti-inflammatory CD4+ T cells in the dorsal root ganglion of female mice.

Chemotherapy is often dose limiting due to the emergence of a debilitating neuropathy. IL-10 and IL-4 are protective against peripheral neuropathy, yet the contribution by CD4+ T cells is unknown. Using flow cytometry, we found that naïve females had a greater frequency of anti-inflammatory CD4+ T cells in the dorsal root ganglion (DRG) compared to males. In response to paclitaxel, females had reduced mechanical hypersensitivity and a greater frequency of anti-inflammatory CD4+ T cells (FoxP3, IL-10, IL-4) in the DRG than male and ovariectomized female mice. These findings support a model in which estrogen promotes anti-inflammatory CD4+ T cells in female DRG to suppress peripheral neuropathy.

Optogenetic Inhibition of Nav1.8 Expressing Corneal Afferents Reduces Persistent Dry Eye Pain.

Purpose: The aim of the present study was to investigate the contribution of Nav1.8 expressing corneal afferent neurons to the presence of ongoing pain in lacrimal gland excision (LGE)-induced dry eye. Methods: The proton pump archaerhodopsin-3/eGFP (ArchT/eGFP) was conditionally expressed in corneal afferents using Nav1.8-cre mice. Dry eye was produced by unilateral LGE. Real time place preference was assessed using a three-chamber apparatus. A neutral, unlit center chamber was flanked by one illuminated with a control light and one illuminated with an ArchT activating light. For real-time preference, animals were placed in the neutral chamber and tracked over five 10-minute sessions, with the lights turned on during the second and fourth sessions. In other studies, movement was tracked over three 10-minute sessions (the lights turned on only during the second session), with animals tested once per day over the course of 4 days. A local anesthetic was used to examine the role of ongoing corneal afferent activity in producing place preference. Results: The corneal afferent nerves and trigeminal ganglion cell bodies showed a robust eGFP signal in Nav1.8-cre;ArchT/eGFP mice. After LGE, Nav1.8-cre;ArchT/eGFP mice demonstrated a preference for the ArchT activating light paired chamber. Preference was prevented with pre-application to the cornea of a local anesthetic. Nav1.8-cre;ArchT/eGFP mice with sham surgery and LGE wild-type control mice did not develop preference. Conclusions: Results indicate LGE-induced persistent, ongoing pain, driven by Nav1.8 expressing corneal afferents. Inhibition of these neurons represents a potential strategy for treating ongoing dry eye-induced pain.

Lacrimal gland excision in male and female mice causes ocular pain and anxiety-like behaviors.

Lacrimal gland excision (LGE) induced dry eye produces more severe corneal damage in female mice, yet signs of LGE-induced ocular pain and anxiety in male and female mice have not been characterized. Excision of either the extraorbital gland (single LGE), or both the extraorbital and intraorbital glands (double LGE) was performed in male and female C57BL/6J mice to induce moderate and severe dry eye. Ongoing pain was assessed by quantifying palpebral opening and evoked nociceptive responses after corneal application of capsaicin and menthol. The open-field and plus maze were used to assess anxiety. Single LGE caused a reduction in palpebral opening and an increase in capsaicin and menthol-evoked responses only in female mice. Furthermore, single LGE produced signs of increased anxiety in female but not male mice. Overall, female mice appear more susceptible to signs of ocular pain, irritation, and anxiety in response to aqueous tear deficiency.

Evaluation of Corneal Damage After Lacrimal Gland Excision in Male and Female Mice.

Purpose: Lacrimal gland excision (LGE) has been utilized in several studies to model aqueous tear deficiency, yet sex as a biological variable has not been factored in to these reports. This study compared corneal pathology in male and female mice following LGE-induced dry eye. Methods: An LGE of either the extraorbital lacrimal gland (single LGE) or both the extraorbital and intraorbital lacrimal glands (double LGE) was performed in male and female C57BL/6J and Balb/cJ mice to produce dry eye of graded severity. Following excision, tearing was evaluated with phenol red thread, and corneal fluorescein staining was scored to quantify the severity of damage. Corneas were evaluated for apoptosis by the TUNEL assay and for cell proliferation using Ki67 staining. Furthermore, corneas were harvested and analyzed for macrophages via flow cytometry. Results: Baseline tearing levels were similar in male and female mice, and LGE resulted in comparable reductions in tearing with the lowest levels recorded after double LGE. As determined by fluorescein staining, LGE produced more severe damage to the cornea in female C57BL/6J and Balb/cJ mice. Double LGE increased TUNEL and Ki67 staining in the cornea, with greater increases found in female mice. Furthermore, LGE produced a greater increase in the total number of corneal macrophages in female mice. Conclusions: These results indicate that female mice are more susceptible to LGE-induced corneal damage. The mechanisms involved in producing these sex differences still need to be elucidated but may involve increased inflammation and macrophage infiltration.

Progesterone Application to the Rat Forehead Produces Corneal Antinociception.

Purpose: Ocular pain and discomfort are the most defining symptoms of dry eye disease. We determined the ability of topical progesterone to affect corneal sensitivity and brainstem processing of nociceptive inputs. Methods: Progesterone or vehicle gel was applied to the shaved forehead in male Sprague Dawley rats. As a site control, gel also was applied to the cheek on the side contralateral to corneal stimulation. Corneal mechanical thresholds were determined using the Cochet-Bonnet esthesiometer in intact and lacrimal gland excision-induced dry eye animals. Eye wipe behaviors in response to hypertonic saline and capsaicin were examined, and corneal mustard oil-induced c-Fos immunohistochemistry was quantified in the brainstem spinal trigeminal nucleus. Results: Progesterone gel application to the forehead, but not the contralateral cheek, increased corneal mechanical thresholds in intact and lacrimal gland excision animals beginning <30 minutes after treatment. Subcutaneous injection of the local anesthetic bupivacaine into the forehead region before application of progesterone prevented the increase in corneal mechanical thresholds. Furthermore, progesterone decreased capsaicin-evoked eye wipe behavior in intact animals and hypertonic saline evoked eye wipe behavior in dry eye animals. The number of Fos-positive neurons located in the caudal region of the spinal trigeminal nucleus after corneal mustard oil application was reduced in progesterone-treated animals. Conclusions: Results from this study indicate that progesterone, when applied to the forehead, produces analgesia as indicated by increased corneal mechanical thresholds and decreased nociceptive responses to hypertonic saline and capsaicin.

Corneal sensitivity following lacrimal gland excision in the rat.

PURPOSE: Dry eye disease (DED) produces ocular pain and irritation, yet a detailed characterization of ocular sensitivity in a preclinical model of DED is lacking. The aim of the present study was to assess nociceptive behaviors in an aqueous tear deficiency model of DED in the rat. METHODS: Spontaneous blinking, corneal mechanical thresholds, and eye wipe behaviors elicited by hypertonic saline (5.0 M) were examined over a period of 8 weeks following the unilateral excision of either the exorbital lacrimal gland or of the exorbital and infraorbital lacrimal glands, and in sham surgery controls. The effect of topical proparacaine on spontaneous blinking and of systemic morphine (0.5-3.0 mg/kg, subcutaneous [SC]) on spontaneous blinking and eye wipe responses were also examined. RESULTS: Lacrimal gland excision resulted in mechanical hypersensitivity and an increase in spontaneous blinking in the ipsilateral eye over an 8-week period that was more pronounced after infra- and exorbital gland excision. The time spent eye wiping was also enhanced in response to hypertonic saline (5.0 M) at both 1- and 8-week time-points, but only in infra- and exorbital gland excised animals. Morphine attenuated spontaneous blinking, and the response to hypertonic saline in dry eye animals and topical proparacaine application reduced spontaneous blinking down to control levels. CONCLUSIONS: These results indicate that aqueous tear deficiency produces hypersensitivity in the rat cornea. In addition, the increase in spontaneous blinks and their reduction by morphine and topical anesthesia indicate the presence of persistent irritation elicited by the activation of corneal nociceptors.