Colleen M. McDowell

Monitoring retinal morphologic and functional changes in mice following optic nerve crush.

PURPOSE We characterized the morphologic and functional changes in optic nerve crushed mice and evaluated electroretinogram (ERG) responses as tools to monitor retinal ganglion cell (RGC) dysfunction. METHODS We performed optic nerve crush (ONC) unilaterally in adult BALB/cJ mice. The neuronal loss in the RGC layer (GCL) and superior colliculus (SC) was determined by Nissl staining. Retinal thickness was assessed by spectral-domain optical coherence tomography (SD-OCT) imaging. Retinal function was determined by pattern ERG and full-field flash ERG. Responses of pattern ERG, positive scotopic threshold response (pSTR), scotopic oscillatory potentials (OPs), and photopic negative response (PhNR) were analyzed. RESULTS The ONC induced progressive neuronal loss in GCL and contralateral SC starting from 7 and 28 days following ONC, respectively. A linear correlation was observed between combined thickness of nerve fiber layer (NFL), GCL, and inner plexiform layer (IPL) imaged by SD-OCT and cell numbers in GCL. Only half of the normal BALB/cJ mice exhibited pattern ERG response, which was smaller and later compared to C57BL/6J mice. The ONC reduced pattern ERG and pSTR, but the reduction of pattern ERG was earlier than pSTR, preceding the anatomical cell loss in the GCL. The PhNR and scotopic OPs were not affected by ONC. CONCLUSIONS The SD-OCT and ERG can be used to monitor noninvasively retinal morphologic and functional changes induced by ONC. Pattern ERG and pSTR are able to detect early RGC dysfunction, but pattern ERG exhibits higher sensitivity. Our results support the use of these tools in studies using the mouse ONC model.

Mutant human myocilin induces strain specific differences in ocular hypertension and optic nerve damage in mice.

Elevated intraocular pressure (IOP) is a causative risk factor for the development and progression of glaucoma. Glaucomatous mutations in myocilin (MYOC) damage the trabecular meshwork and elevate IOP in humans and in mice. Animal models of glaucoma are important to discover and better understand molecular pathogenic pathways and to test new glaucoma therapeutics. Although a number of different animal models of glaucoma have been developed and characterized, there are no true models of human primary open angle glaucoma (POAG). The overall goal of this work is to develop the first inducible mouse model of POAG using a human POAG relevant transgene (i.e. mutant MYOC) expression in mouse eyes to elevate IOP and cause pressure-induced damage to the optic nerve. Four mouse strains (A/J, BALB/cJ, C57BL/6J, and C3H/HeJ) were used in this study. Ad5.MYOC.Y437H (5 × 10(7) pfu) was injected intravitreally into one eye, with the uninjected contralateral eye serving as the control eye. Conscious IOP measurements were taken using a TonoLab rebound tonometer. Optic nerve damage was determined by scoring PPD stained optic nerve cross sections. Retinal ganglion cell and superior colliculus damage was assessed by Nissl stain cell counts. Intravitreal administration of viral vector Ad5.MYOC.Y437H caused a prolonged, reproducible, and statistically significant IOP elevation in BALB/cJ, A/J, and C57BL/6J mice. IOPs increased to approximately 25 mm Hg for 8 weeks (p < 0.0001). In contrast, the C3H/HeJ mouse strain was resistant to Ad5.MYOC.Y437H induced IOP elevation for the 8-week time period. IOPs were stable (12-15 mm Hg) in the uninjected control eyes. We also determined whether there were any strain differences in pressure-induced optic nerve damage. Even though IOP was similarly elevated in three of the strains tested (BALB/cJ, C57BL/6J, and A/J) only the A/J strain had considerable and significant optic nerve damage at the end of 8 weeks with optic nerve damage score of 2.64 ± 0.19 (n = 18, p < 0.001) in the injected eye. There was no statistical difference in retinal ganglion cell death or superior colliculus damage at the 8-week time point in any of the strains tested. These results demonstrate strain dependent responses to Ad5.MYOC.Y437H-induced ocular hypertension and pressure-induced optic nerve damage.

Optic nerve crush induces spatial and temporal gene expression patterns in retina and optic nerve of BALB/cJ mice

BackgroundCentral nervous system (CNS) trauma and neurodegenerative disorders trigger a cascade of cellular and molecular events resulting in neuronal apoptosis and regenerative failure. The pathogenic mechanisms and gene expression changes associated with these detrimental events can be effectively studied using a rodent optic nerve crush (ONC) model. The purpose of this study was to use a mouse ONC model to: (a) evaluate changes in retina and optic nerve (ON) gene expression, (b) identify neurodegenerative pathogenic pathways and (c) discover potential new therapeutic targets.ResultsOnly 54% of total neurons survived in the ganglion cell layer (GCL) 28 days post crush. Using Bayesian Estimation of Temporal Regulation (BETR) gene expression analysis, we identified significantly altered expression of 1,723 and 2,110 genes in the retina and ON, respectively. Meta-analysis of altered gene expression (≥1.5, ≤-1.5, p < 0.05) using Partek and DAVID demonstrated 28 up and 20 down-regulated retinal gene clusters and 57 up and 41 down-regulated optic nerve clusters. Regulated gene clusters included regenerative change, synaptic plasticity, axonogenesis, neuron projection, and neuron differentiation. Expression of selected genes (Vsnl1, Syt1, Synpr and Nrn1) from retinal and ON neuronal clusters were quantitatively and qualitatively examined for their relation to axonal neurodegeneration by immunohistochemistry and qRT-PCR.ConclusionA number of detrimental gene expression changes occur that contribute to trauma-induced neurodegeneration after injury to ON axons. Nrn1 (synaptic plasticity gene), Synpr and Syt1 (synaptic vesicle fusion genes), and Vsnl1 (neuron differentiation associated gene) were a few of the potentially unique genes identified that were down-regulated spatially and temporally in our rodent ONC model. Bioinformatic meta-analysis identified significant tissue-specific and time-dependent gene clusters associated with regenerative changes, synaptic plasticity, axonogenesis, neuron projection, and neuron differentiation. These ONC induced neuronal loss and regenerative failure associated clusters can be extrapolated to changes occurring in other forms of CNS trauma or in clinical neurodegenerative pathological settings. In conclusion, this study identified potential therapeutic targets to address two key mechanisms of CNS trauma and neurodegeneration: neuronal loss and regenerative failure.

Gremlin Induces Ocular Hypertension in Mice Through Smad3-Dependent Signaling

PURPOSE Transforming growth factor-β2 induces extracellular matrix (ECM) remodeling, which likely contributes to the defective function of the trabecular meshwork (TM) leading to glaucomatous ocular hypertension. Bone morphogenetic proteins (BMPs) inhibit these profibrotic effects of TGFβ2. The BMP antagonist gremlin is elevated in glaucomatous TM cells and increases IOP in an ex vivo perfusion culture model. The purpose of this study was to determine whether gremlin regulates ECM proteins in the TM, signals through the Smad3-dependent pathway, and induces ocular hypertension in mice. METHODS Ad5.Gremlin or Ad5.TGFβ2 was injected intravitreally into one eye of each mouse. Intraocular pressure measurements were taken using a TonoLab tonometer. Gremlin, TGFβ2, fibronectin (FN), and collagen-1 (Col-1) expression in the TM was determined by immunofluorescence, Western immunoblot, and quantitative (q)PCR analyses. RESULTS Ad5.Gremlin or Ad5.TGFβ2 each caused significant IOP elevation in mice. Immunofluorescence and Western blot analysis demonstrated that gremlin and TGFβ2 reciprocally increased the expression of each other, and both increased FN expression in the TM and surrounding tissues. Ad5.Gremlin elevated IOP and increased Fn and Col-1 gene expression in the TM of Smad3 wild-type (WT) mice, but had no effect in Smad3 HET or Smad3 KO mice. CONCLUSIONS Our results demonstrate that intravitreal injections of either Ad5.Gremlin or Ad5.TGFβ2 elevate IOP and upregulate the ECM protein FN in the TM of mice. These data show that gremlin signals through the Smad3-dependent pathway in the TM to elevate IOP. We determined for the first time gremlins role in inducing ocular hypertension in an in vivo model system.

Crosstalk Between Transforming Growth Factor Beta-2 and Toll-Like Receptor 4 in the Trabecular Meshwork

Purpose The trabecular meshwork (TM) is involved in the outflow of aqueous humor and intraocular pressure (IOP) regulation. Regulation of the extracellular matrix (ECM) by TGFβ2 signaling pathways in the TM has been extensively studied. Recent evidence has implicated toll-like receptor 4 (TLR4) in the regulation of ECM and fibrogenesis in liver, kidney, lung, and skin. Here, we investigated the role of TGFβ2–TLR4 signaling crosstalk in the regulation of the ECM in the TM and ocular hypertension. Methods Cross sections of human donor eyes, primary human TM cells in culture, and dissected mouse TM rings were used to determine Tlr4 expression in the TM. Trabecular meshwork cells in culture were treated with TGFβ2 (5 ng/mL), TLR4 inhibitor (TAK-242, 15 μM), and a TLR4 ligand (cellular fibronectin isoform [cFN]-EDA). A/J (n = 13), AKR/J (n = 7), BALBc/J (n = 8), C3H/HeJ (n = 20), and C3H/HeOuJ (n = 10) mice were injected intravitreally with adenovirus 5 (Ad5).hTGFβ2c226s/c228s in one eye, with the uninjected contralateral eye serving as a control. Conscious IOP measurements were taken using a TonoLab rebound tonometer. Results Toll-like receptor 4 is expressed in the human and mouse TM. Inhibition of TLR4 signaling in the presence of TGFβ2 decreases fibronectin expression. Activation of TLR4 by cFN-EDA in the presence of TGFβ2 further increases fibronectin, laminin, and collagen-1 expression, and TLR4 signaling inhibition blocks this effect. Ad5.hTGFβ2c226s/c228s induces ocular hypertension in wild-type mice but has no effect in Tlr4 mutant (C3H/HeJ) mice. Conclusions These studies identify TGFβ2–TLR4 crosstalk as a novel pathway involved in ECM regulation in the TM and ocular hypertension. These data further explain the complex mechanisms involved in the development of glaucomatous TM damage.

BMP and Activin Membrane Bound Inhibitor Regulates the Extracellular Matrix in the Trabecular Meshwork

Purpose The trabecular meshwork (TM) has an important role in the regulation of aqueous humor outflow and IOP. Regulation of the extracellular matrix (ECM) by TGFβ2 has been studied extensively. Bone morphogenetic protein (BMP) and activin membrane-bound inhibitor (BAMBI) has been shown to inhibit or modulate TGFβ2 signaling. We investigate the role of TGFβ2 and BAMBI in the regulation of TM ECM and ocular hypertension. Methods Mouse TM (MTM) cells were isolated from B6;129S1-Bambitm1Jian/J flox mice, characterized for TGFβ2 and dexamethasone (DEX)–induced expression of fibronectin, collagen-1, collagen-4, laminin, α-smooth muscle actin, cross-linked actin networks (CLANs) formation, and DEX-induced myocilin (MYOC) expression. MTM cells were transduced with Ad5.GFP to identify transduction efficiency. MTM cells and mouse eyes were transduced with Ad5.Null, Ad5.Cre, Ad5.TGFβ2, or Ad5.TGFβ2 + Ad5.Cre to evaluate the effect on ECM production, IOP, and outflow facility. Results MTM cells express TM markers and respond to DEX and TGFβ2. Ad5.GFP at 100 MOI had the highest transduction efficiency. Bambi knockdown by Ad5.Cre and Ad5.TGFβ2 increased fibronectin, collagen-1, and collagen-4 in TM cells in culture and tissue. Ad5.Cre, Ad5.TGFβ2, and Ad5.TGFβ2 + Ad5.Cre each significantly induced ocular hypertension and lowered aqueous humor outflow facility in transduced eyes. Conclusions We show for the first time to our knowledge that knockdown of Bambi alters ECM expression in cultured cells and mouse TM, reduces outflow facility, and causes ocular hypertension. These data provide a novel insight into the development of glaucomatous TM damage and identify BAMBI as an important regulator of TM ECM and ocular hypertension.

Subtype-specific response of retinal ganglion cells to optic nerve crush

Glaucoma is a neurodegenerative disease with retinal ganglion cell (RGC) loss, optic nerve degeneration and subsequent vision loss. There are about 30 different subtypes of RGCs whose response to glaucomatous injury is not well characterized. The purpose of this study was to evaluate the response of 4 RGC subtypes in a mouse model of optic nerve crush (ONC). In this study, we also evaluated the pattern of axonal degeneration in RGC subtypes after nerve injury. We found that out of the 4 subtypes, transient-Off α RGCs are the most susceptible to injury followed by On–Off direction selective RGCs (DSGC). Non-image forming RGCs are more resilient with ipRGCs exhibiting the most resistance of them all. In contrast, axons degenerate irrespective of their retinal soma after ONC injury. In conclusion, we show that RGCs have subtype specific cell death response to ONC injury and that RGC axons disintegrate in an autonomous fashion undergoing Wallerian degeneration. These discoveries can further direct us towards effective diagnostic and therapeutic approaches to treat optic neuropathies, such as glaucoma.