The impact of obesity; key features of raised intracranial pressure and clinical sequalae in rodents

Elevated intracranial pressure (ICP) is observed in many brain disorders. Obesity has been linked to ICP pathogenesis in disorders such as idiopathic intracranial pressure (IIH). We investigated the effect of diet induced obesity (DIO) on ICP and clinically relevant sequelae. Rats were fed either a control or high fat diet. Following weight gain long term ICP, headache behavior, body composition and retinal outcome were examined. Post-hoc analysis of retinal histology and molecular analysis of choroid plexus and trigeminal ganglion (TG) were performed. DIO rats demonstrated raised ICP by 55% which correlated with the abdominal fat percentage and increased non-respiratory slow waves, suggestive of altered cerebral compliance. Concurrently, DIO rats demonstrated a specific cephalic cutaneous allodynia which negatively correlated with the abdominal fat percentage. This sensitivity was associated with increased expression of headache markers in TG. Additionally, DIO rats had an in vivo increased retinal nerve fiber layer thickness associated with raised ICP with a subsequent post-hoc demonstration of neuroretinal degeneration. This study demonstrates for the first time that DIO leads to raised ICP and subsequent clinically relevant symptom development. This novel model of non-traumatic raised ICP could expand the knowledge regarding disorders with elevated ICP such as IIH.

Rats were fed either a control or high fat diet. Following weight gain long term ICP, headache 23 behavior, body composition and retinal outcome were examined. Post-hoc analysis of retinal 24 histology and molecular analysis of choroid plexus and trigeminal ganglion (TG) were performed. 25 DIO rats demonstrated raised ICP by 55% which correlated with the abdominal fat percentage and 26 increased non-respiratory slow waves, suggestive of altered cerebral compliance. Concurrently, DIO 27 rats demonstrated a specific cephalic cutaneous allodynia which negatively correlated with the 28 abdominal fat percentage. This sensitivity was associated with increased expression of headache 29 markers in TG. Additionally, DIO rats had an in vivo increased retinal nerve fiber layer thickness 30 associated with raised ICP with a subsequent post-hoc demonstration of neuroretinal degeneration. 31 This study demonstrates for the first time that DIO leads to raised ICP and subsequent clinically 32 relevant symptom development. This novel model of non-traumatic raised ICP could expand the 33 knowledge regarding disorders with elevated ICP such as IIH.

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Elevation in intracranial pressure (ICP) is observed in a range of cerebral pathologies, such as traumatic 38 brain injury (TBI), ischemic stroke, hydrocephalus and idiopathic intracranial hypertension (IIH). 39 These disorders are associated with significant morbidity and are amongst the most debilitating, costly 40 brain disorders 1 . Currently, limited pharmacotherapies exist to treat patients with elevated ICP, as the aquaporin water channels (AQPs) and water co-transporting proteins as the Na + -K + -2Cl --1 45 cotransporter (NKCC1) have improved our understanding of this process 2 . 46 The normal range of ICP in healthy adults is around -2 to 5 mmHg and is influenced by body positions, 47 aging and diurnal variations 3-5 . In recent years, studies have suggested that obesity may also be 48 associated with ICP dynamics indicated by an association between elevated BMI and ICP 6,7 . In 49 support, a direct correlation between BMI, percentage body fat and CSF pressure has been 50 demonstrated 8 . Furthermore, obesity is the major risk factor for development of IIH, which is a 51 metabolic disease of increased ICP without identifiable cause. The condition affects mainly women and 52 90% of the patients are obese 9 . IIH has significant morbidity and the most common symptoms are 53 chronic headache, cognitive impairment and impaired vision due to papilledema 10,11 . The severity of 54 papilledema and visual loss is associated with increasing BMI in IIH 12,13 . Headache is also associated 55 with other raised ICP disorders such as TBI and hydrocephalus. Obesity is also associated with 56 migraine and is a risk factor for chronic headache 14,15 . The nature of IIH, with its atraumatic intracranial hypertension, chronic headache and impaired vision 58 renders IIH an appealing model condition of elevated ICP that could provide valuable new knowledge 59 regarding both regulation and dysregulation of ICP dynamics in pathology. However, no proper 60 preclinical model for non-traumatic raised ICP or IIH exists. Our group developed the first rodent 61 model mimicking aspects of obesity-induced raised ICP using Zucker rats (genetic model of obesity) 16 . 62 However, the increasing rate of obesity suggests that environmental and behavioral factors including 63 dietary factors have been the major contributors to the obesity epidemic rather than rare genetic 64 changes 17 . There is no evidence that IIH is a genetic disease, as such the source of the disease is likely 65 behavioral and environmental. Therefore, we have used DIO rats in this study which share many 66 characteristics with the common form of human obesity. The aim was to explore the relationship 67 between DIO, raised ICP, headache and structural morphology of the optic nerve head (ONH) and 68 neuroretina using digital high-resolution and image-guided spectral domain optical coherence 69 tomography (OCT). A novel and validated telemetric system to monitor ICP was used 18 Fig. 1M). This loss of significance in ICP could be 94 6 mediated by weight loss. Although both groups lost weight as expected, DIO rats lost more weight 95 before regaining weight to a lesser amount (-7.5±0.6% vs 0.6±1.7%, P=0.0004, Fig. 1N) 18,19 .

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To further interrogate the headache phenotype, we assessed gene expression in TG on day 30 (Fig. 3F).

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In the DIO rats, there was a 1.24 fold increase in Calca (CGRPα) expression (2.9±0.05 vs 3.41±0.15,  (Table 1). No significant thickness 157 difference between DIO and control animals was found for TR, GCC, IRL and ORL (Table 1)     IIH is a challenging disease with increased ICP of unknown etiology and is also closely linked to 221 obesity. The incidence is rapidly increasing in the wake of the global obesity epidemic 9 . Importantly, a 222 recent study demonstrated that in non-IIH patients, BMI and percentage body fat both positively 223 correlated with CSF opening pressure, although to a lesser degree than seen in IIH 8 . In support, we 224 have recently shown that with normal weight gain in rats, there was a trend to an increase in ICP, 225 supporting the idea of obesity has an impact on ICP 18 . In the current study, raised ICP was found in the 226 DIO group, where the ICP was raised by 55% and associated with increased abdominal obesity. This 227 study is congruent with our previous study in which it was shown that genetic obesity in rats caused 228 40% higher ICP 16 . As in our previous study using the same telemetric system for ICP monitoring in 229 normal rats, the resting ICP value for the rats at the day of surgery increased steadily on the following 230 days and stabilized around day 7 18 . Previously, we demonstrated that ICP dynamics are influenced by 231 recovery from surgery and the use of anesthesia alters the ICP waveforms 21 . Interestingly, it was found 232 that the DIO rats had increased non-respiratory slow waves after recovery from surgery, potentially 233 indicating an altered cerebral compliance.

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It has been demonstrated that female obese rats have increased CSF secretion 23 . In the current non-248 traumatic raised ICP model, there was a trend to increased mRNA expression of Aqp1 and Slc12a2. 249 This trend was also observed in protein expression, where there was tendency to increased ratio of 250 glycosylated AQP1 to total AQP1. Due to a slower weight gain after surgery in the DIO rats, the 251 weight difference at 30 days was decreased to 3.4%. This may have affected the outcome in the 252 19 molecular analysis and therefore requires further investigations. We have previously shown that obese 253 Zucker rats with elevated ICP had increased expression of AQP1 at CP 16 . Furthermore, AQP1 254 knockouts had 56% lower ICP in comparison with wild-type mice 24 . All together this suggests that the 255 obesity-induced ICP may modulate CSF dynamics by altering the expression of the water channels.

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Headache is a frequent symptom in pathologies with raised ICP such as TBI, hydrocephalus and IIH 25 .

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Headache is present in around 75-94% of IIH patients at diagnosis [26][27][28] . In the present study, we show 258 that DIO rats had cephalic cutaneous mechanical allodynia but not peripheral, where the severity was    Severe obesity is found to be related to retinal neurodegeneration 42 . In this study, the OCT data 294 showed thickening of the peripapillary RNFL in DIO rats with a significant positive correlation to ICP.

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Across both the DIO and the control animals, there was also a clear correlation between RNFL 296 thickening and the total body weight and as well the abdominal fat percentage. This identifies 297 abdominal obesity as a potentially disease aggravating factor. We suggest this in vivo RNFL thickening 298 observed with OCT may be due to swelling of the retinal ganglion cell axons due to axoplasmic flow 299 stasis. This initial RNFL swelling is also present in other models with varying optic nerve or disc 300 damage 43,44 . By histology, we showed that after a prolonged period of DIO and elevated ICP there was 301 a significant thinning of RNFBs entering the optic disc. This is in accordance with the clinical setting 302 where obesity is found to correlate with both raised ICP and RNFL thinning 42,45 . Furthermore, we 303 demonstrate that the degree of RNFL swelling at OCT is negatively associated with RNFB thickness at     The ODP images were evaluated for obvious pathology including optic disc obscuration, peripapillary 400 halo, vessel engorgement/torsion/obscurations, hemorrhages and choroidal folds. Intraocular pressure 401 (IOP) was measured in non-anaesthetized rats on day 0 using a TONOLAB tonometer (ICARE, 402 Finland). The investigator was blinded to both the experimental group and identity of the rat. Blood glucose was measured on day 30, where the rats had been fasted overnight for at least 14h.

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Blood from the lateral tail vein was used to asses blood glucose (mmol/L) using glucometer ACCU-