**3. Ascorbic Acid Transport and Synthesis: Animal Models**

Early work on AA and the effects of deficiency was accomplished using guinea pigs that are among the few non-primate mammals that naturally lack a functional *gulo* gene [77]. These studies on the course and effects of scurvy were conducted even before the identification of ascorbic acid as vitamin C [78,79] with inclusion of "the antiscorbutic factor" [80], (such as in fresh raw cabbage). Guinea pigs are particularly sensitive to AA (and other) deficiencies [81,82], and are still a highly utilized model for a number of human disease states, for example in relation to dyslipidemia (reviewed in [83]). The recent (since 2000) increased focus on mouse models results from the ability to use genetically altered mouse lines to impact both transport and synthesis in the same animals. Much more is known about the mouse genome than guinea pig and in many cases the models have been also bred to the same background strain making experiments directly comparable. Data can thus be interpreted, and experiments planned, with reference to the huge literature of biomedical and pharmaceutical research that has already taken place in mice making disease and treatment-relevant findings much more likely. Brief descriptions of the relevant mouse models, including gulo knockout mice, and SVCT1/2 knockout models and SVCT2 transgenic mice are reported hereafter.

Gulo knockout mice: Gulo knockout mice carry an inactivated form of the gene L-gulono-γ-lactone oxidase and are thus completely dependent on dietary AA [84]. These mice, when maintained on

sub-optimal supplementation levels, are the closest to the human state of lifetime AA dietary deficiency. Knockout mice fed low levels of AA have decreased antioxidant capacity and also altered cholesterol metabolism and exhibit profound changes in vasculature. Oxidative stress has since been confirmed in these mice in adults and during development [11,85,86]. These mice also have an aberrant behavioral phenotype including poorer sensorimotor skills, and altered response to activity-inducing effects of cholinergic and dopaminergic compounds scopolamine and methamphetamine even when adequately supplemented as adults [86–88]. It is proposed that these changes result from low AA during some part of the developmental process. AA deficient gulo mice have been the subject of metabolomics profiling which has identified metabolic shifts as a response to oxidative stress of AA deficiency (e.g., upregulation of glutathione production, decreased carnitine production) [89]. Neutrophils from AA deficient gulo mice are more likely to undergo necrosis than normal apoptosis. They are not recognized by macrophages and thus clearance is also affected [90]. Such responses are important in any disease where inflammation is a factor, such as AD. Senescence marker protein 30 (SMP30) was first identified as a protein that decreased with age. Sequencing and creation of an SMP30 knockout mouse later identified this protein as the gulo enzyme [91,92]. These mice are likewise dependent on dietary AA and under conditions of deficiency their brains are susceptible to oxidative stress (generation of superoxide) [93,94]. A further mouse line exhibiting spontaneous bone fracture derived from in-bred balb/c mice [95] does not synthesize AA [96]. These mice die at an early age if not supplemented, and have been used for studies of AA level and gene transcription [97], however, this line is much less well-described. Disruption to catecholamine levels in brain and adrenal glands has been shown in each of these mouse lines [98,99]. Such data that follow a consistent pattern across the different mouse models are a strong support for the role of AA in any particular area and also of the validity of the models themselves.

SVCT1 knockout mice: SVCT1 knockout mice are viable and fertile [49]. They excrete significantly more AA in the urine than wild type and heterozygous littermates and have lower circulating blood levels. In addition to this loss of renal reabsorption of AA, uptake into liver was also dramatically decreased. Also of interest is the lower brain levels in these mice, presumably linked to lower circulating levels because SVCT1 is not expressed in brain and so should not have a direct effect (Figure 3).

SVCT2 knockout mice: The first report of the SVCT2 knockout mouse described homozygous mice that did not survive past birth (newborns never take a first breath) and had almost undetectable AA levels in all organs measured (brain, liver, adrenal glands, kidney, pituitary glands, pancreas and muscle) [100]. Subsequent studies found that AA levels were also decreased in placenta and lung as well as brain and other organs reported previously [101]. These low AA levels were associated with increased oxidative stress and cell death. Hemorrhaging, initially only reported in cortical areas [100] was also seen in the brain stem area which may contribute to the lack of breathing in the newborn mice, particularly because no obvious problems were identified in the lung in the earlier study. Primary cultured cells from hippocampi of embryonic SVCT2 knockout mice showed decreased neuronal activity, stunted neurite outgrowth and were much more sensitive to exogenously administered oxidative stress [72]. Investigation into catecholamine systems in these mice has yielded mixed data. One report showed large decreases (50%) of norepinephrine in the adrenal glands with no change in dopamine levels, whereas smaller changes in the brain (decrease of 20%–25%) were not significant

for either dopamine or norepinephrine in SVCT2 knockout mice [102]. Additionally, morphological differences in the chromaffin cells of the adrenal glands were also noted in this study. Other reports have shown significant alteration to dopamine and norepinephrine systems, including metabolites, in embryonic SVCT2 knockout and SVCT2 transgenic (described below) brains [103].

Increase in AA in SVCT2-Tg mice is dependent on level of over-expression, which varies among mice and in this small sample was only 1.3-2.0-fold compared to wild-type (not shown). Brain levels did not differ between supplemented (black triangles) and non-supplemented (grey triangles) SVCT1 knockout mice so combined average is shown. Normal brain levels can be achieved in gulo knockout mice with supplementation of at least 0.33 g/L, (shown here at supplementation with 1.0 g/L). Mean AA levels that share a superscript do not differ. Differing superscripts indicate significant difference (a, b) *p* < 0.05, (c, d) *p* < 0.001 by Univariate ANOVA with Tukey *post-hoc* comparisons (overall group effect F (4,18) = 13.0, *p* < 0.001).

SVCT2 transgenic mice: Recently, a new mouse line has been created that expresses additional copies of the SVCT2 [104]. This mouse has increased SVCT2 mRNA expression in all organs measured and a related increase in AA level in all organs except the liver, with up to two-fold increases in the brain, depending on mRNA expression. These mice were otherwise phenotypically indistinguishable from wild type but were more resistant to a severe oxidative stress in lung induced by paraquat. Although not designed to represent an attainable human situation, these mice may be able to inform on SVCT2 regulation and the role of vitamin C in high oxidative stress situations such as neurodegenerative disease.
