**4.4 Normotensive glaucoma models**

Genetically modified mice with normal IOP have been used to further study normotensive glaucoma. Specifically, mice lacking the glutamate transporter genes, *Glast* or *Eaac1* (Excitatory amino acid transporter 3), develop RGC damage and optic nerve degeneration without the IOP elevation. *Glast* is expressed in Müller glia in the retina and aids in the removal of glutamate, sparing RGCs from the neuroexcitotoxicity due to its accumulation [46]. *Eaac1* is expressed in neurons and assists with the uptake of both cysteine and glutamate [46]. This evidence supports a protective role of these genes in preventing RGC damage.

A deficiency in apoptosis signal-regulating kinase 1 (*Ask1*) also known as mitogen-activated protein kinase 5 (Map3k5) had no neurotoxic effects or effects on IOP. It was also demonstrated that the activation of p38 MAPK and the production of inducible nitric oxide synthase was suppressed in glial cells and RGCs, implying that ASK1 activation could be involved in normotensive glaucoma [67]. Additionally, this model was used to study the neuroprotective effects of *N*-acetylcysteine, which was demonstrated to be protective in the *Eaac1* KO mice. Kimura *et al*. also added that *Glast* or *Eaac1* mutations produce an early onset phenotype, which increases the utility of this model, particularly in studying efficacy of treatments [46]. This is contrasted with the other models of normotensive glaucoma, which are comprised of overexpression of mutated optineurin E50K and tank-binding protein 1, the mutated genes associated with human normotensive glaucoma [46]. These models have a later onset, which makes them less suitable experimentally. In addition, using marmosets mentioned prior, a model is in the works targeting the *Glast* gene, which is speculated to produce a functional model with early onset glaucoma [46].

### **4.5 Immune response models**

In the Wallerian degeneration slow (W1dS) mouse, transfection with a fusion gene of *Nmnat1* and *Ube4b* protect RGC axons by maintaining mitochondrial function. An influx of calcium was documented prior to RGC axon degeneration [68, 69]. This finding was significant, as it proves there are specific molecular mechanisms that modulate axon degeneration. Struebing *et. al* review other molecular mechanisms of RGC degeneration. For example, knocking out *Bax* promotes survival of

RGC bodies, but does not rescue axons. The JNK (c-Jun, N-terminal kinase) pathway is involved in both axonal and soma degradation as deficiency in both *Jnk2* and *Jnk3* results in protection from RGC death after optic nerve crush. Lastly, knocking out *Dlk* (dual leucine zipper kinase) causes cell death in soma but does not affect degeneration of axons [20, 70–72].

Within an investigation of an autoimmune response as a plausible mechanism of RGC damage, it was determined that serum samples of glaucoma patients have increased levels of heat shock protein 27 (HSP27) and heat shock protein 60 (HSP60). Immunization of these proteins in the Lewis rat population resulted in RGC degeneration and axonal loss 1–4 months later, supporting a role of these proteins in glaucoma [11, 73].

#### **4.6 The BXD murine family of recombinant inbred lines of mice**

Because most human glaucoma cases are not due to polymorphisms in single genes, it is not surprising that pre-clinical models that examine the effects of mutations in one gene at a time do not fully recapitulate all endophenotypes of human disease. In contrast, a polygenetic approach may advance the field of glaucoma. One approach to this is the involvement of the BXD murine family. BXD mice are a family of recombinant inbred strains that were derived by mating C57/Bl6 (B6) and DBA/2 J (D2) mice and inbreeding the F2 progeny for >20 generations, allowing for natural recombination of genes throughout the entire genome.

Each fully inbred BXD strain is genetically distinct and fixed at each locus of the genome. This family of mice is an outstanding resource as data on >1000 phenotypes and nearly 100 expression data sets (gene, protein, and metabolites) have been collected and are available on GeneNetwork (www.genenetwork.org), an open access resource for the scientific community. Moreover, all BXD and their parent strains have been fully sequenced. There are ~5.2 million single nucleotide polymorphisms, >400,000 insertions/deletions and multiple copy number variants between the progenitors which segregate among the BXD strains. Multiple data sets of the eye, retina, optic nerve phenotypes and expression data are also available on GeneNetwork to facilitate systems genetics analyses at multiple levels from gene to metabolome [74].

The BXD family has become a valuable resource for modeling human disease, including glaucoma. B6 has no glaucoma-associated pathologies with fairly constant IOP until >13 months of age. The optic nerve has little damage throughout the life of B6. In contrast, D2 parents develop pigmentary dispersion glaucoma and have an elevated IOP that reaches its maximum at ~9 months with a subsequent IOP reduction. ON damage increases throughout the lifetime of D2 mice. The influence of pigmentary dispersion on glaucoma can be studied among the afflicted progeny or be eliminated by excluding those strains that harbor mutations in *Tyrp1* and *Gpnmb* from the study plan. By quantifying specific endophenotypes across the BXD family along with gene expression and polymorphism profiles, new and informative insights regarding novel modulating genes can be revealed. Specific to this pre-clinical murine family, several BXD strains spontaneously develop elevated IOP and/or optic nerve damage due to the particular set of polymorphisms in the genome of each individual strain, similar to the human condition. Importantly, the phenotype of each BXD strain is highly reproducible because each strain is fully inbred with a fixed genome and the supply of mice from each strain is essentially unlimited.

As a testament to the power of the BXD family and the genetic/genomic information gathered from these mice, approximately 20 disease-associated strains have already been cloned from the BXD family [74]. One example of the power of using BXD mice in the virtuous cycle of bi-directional translation is the identification of

*An Overview of Glaucoma: Bidirectional Translation between Humans and Pre-Clinical… DOI: http://dx.doi.org/10.5772/intechopen.97145*

the novel IOP-modulating gene, *Cacna2d1*, which has demonstrated potent IOPlowering effects with therapeutic blocking of the function of its gene product with pregabalin in a dose-dependent and haplotype-specific manner [74].

In other studies, by examining the immune network and the response of the optic nerve to RGC damage, and comparing the two, it was determined that an innate immune network was activated by the optic nerve crush [75]. The importance of the complement cascade or innate immune network as mentioned above, has been demonstrated by knocking out *C1qa* in D2 mice. Both pigmentary dispersion glaucoma and elevated IOP are noted, however, the loss of axons in the optic nerve is lessened, which points to the role of *C1qa* in the degeneration of axons in the optic nerve [76]. A gene network of regulatory mechanisms in the retina that are activated by injury was mapped, and it was determined there are common regulatory mechanisms. In addition, there was a significant quantitative trait locus (QTL) found on chromosome 16 from 80 to 95 Mb that correlates with the expression of *C4b* in the normal retina [77]. In this region, two *cis*-acting QTLs were identified as potential modulatory genes of the innate immunity network, although the modulating gene has not yet been identified [20]. These data suggest possible shared disease mechanisms with age-related macular degeneration (AMD), in which complement proteins are found in the drusen deposits that characterize AMD [45, 78–80].
