Life Sci. 2016 Apr 15;151:15-22.

Adjuvant-induced mono-arthritis potentiates cerebral hemorrhage in the spontaneously hypertensive rats with increased severity due to high salt diet.

Amy Randell*, and Noriko Daneshtalab*

300 Prince Philip Drive, Health Sciences Center, Memorial University of Newfoundland, School of Pharmacy, St. John’s Newfoundland, CANADA A1B 3V6

 

Abstract

Aims: Patients with rheumatoid arthritis (RA), have a higher incidence of hypertension and stroke than the normal population. Currently there exists no animal model to study the pathogenic interactions of hemorrhagic stroke (HS) subsequent to chronic inflammation and hypertension. We have created and defined a hypertensive-mono-arthritic animal model who demonstrate gros signs of cerebral hemorrhage in presence of mono-arthritis.

Main Methods: Spontaneously hypertensive rats (SHR) were fed either a high salt diet (4% NaCl; HSD) or Purina chow (RD) from weaning. Complete Freund’s adjuvant (CFA) was injected into the left hind paw at 21-28 weeks (Control groups received saline (SAL)). Degree of inflammation, joint swelling, weight and blood pressure were monitored for 21 days. Animals were then sacrificed and their brain and left hind paw evaluated.

Key Findings: All groups were hypertensive throughout the experimental period (>180mmHg systolic), irrespective of diet. Both CFA groups produced significant local inflammatory response in their injected paw with associated joint degradation and cellular infiltrates. Systemic plasma TNF-α levels were significantly elevated in CFA groups, with significant increase in TNF-α at 7 and 14 days, compared to SAL groups. Cerebral hemorrhage was visualized in the CFA groups but not SAL controls, with a higher severity in HSD-CFA group.

Significance: The mono-arthritic hypertensive animals are capable of developing HS upon induction of inflammatory insult. The HSD appears to exacerbate the inflammatory response and influence degree of the hemorrhage. Our novel, multi-disease model may provide an appropriate platform to study the pathogenesis of HS among arthritic patients.

KEYWORDS: Adjuvant-induced-arthritis; Animal models; Complete Freund’s adjuvant; Hemorrhagic stroke; High salt diet; Hypertension; Mono-arthritis; Rheumatoid arthritis; Spontaneously hypertensive rats

PMID: 26903291

 

Supplement

Our research program studies the cardiovascular pathogenesis associated with autoimmune disease, particularly pertaining to hypertension and arthritis/systemic inflammation and the risk of stroke development. Patients with autoimmune disease, such as rheumatoid arthritis (RA), have a higher incidence of hypertension and stroke than the normal population. Hypertension itself is a highest risk factor for stroke and heart attack and is the cause of middle cerebral artery failure, causing microhemorrhages in the brain. We study the link between hypertension and RA in the development of stroke.

 

When it comes to stroke, ischemic stroke is more common, and is often more easily treatable with drugs. The second type of stroke is due to bleeding in the brain (hemorrhagic stroke) rather than ischemic stroke, because of blood leakage into the brain. Although it occurs less often than ischemic stroke (about 15-20% of strokes are attributed to hemorrhagic stroke), it is the more deadly, with 40% of deaths associated with hemorrhagic stroke. A large issue is that there is no pharmacological treatment associated with hemorrhagic stroke, with only surgical intervention. Finding the reason behind what causes hemorrhagic stroke, and particularly what induces RA patients to have more stroke (both ischemic and hemorrhagic) will be very important in finding an appropriate treatment,

 

Part of the problem leading to hemorrhagic stroke is that it is associated with the loss in the ability of the middle cerebral artery (MCA) of the brain to undergo pressure dependent constriction and maintain constant blood flow, particularly with a high systemic blood pressure (what usually causes stroke).  Because of that, the smaller brain vessels burst and you have bleeding in the brain. Interestingly, hemorrhagic stroke is not associated with inflammation. However, we believe that in arthritic patients, part of the problem that leads to more stroke occurring is the increase in the systemic inflammation in the body which may cause an increase in brain inflammation. We believe it is the action of the inflammatory mediators in the brain that affect the ability of the MCA brain vessels to function properly and constrict in response to pressure.

 

Unfortunately, it was not possible to study this critical issue and phenomenon in brain vessel samples from human patients, and until now, no animal model existed to study the pathogenesis of hemorrhagic stroke subsequent to chronic inflammation and longstanding hypertension. The current article presented here outlines the creation and definition of a hypertensive-mono-arthritic animal model which reflects the physiological conditions associated with mono-arthritis and with high blood pressure. We hypothesized that if we take the spontaneously hypertensive rat (SHR) strain (which become hypertensive but are normally resistant to stroke) and we induced a chronic systemic inflammation, we can induce hemorrhagic stroke formation (Figure 1).

 

The model of chronic systemic inflammation is induced using the injection of Complete Freund’s Adjuvant in one paw and causes physiological symptoms akin to that seen in RA patients with joint swelling, stiffness, local and systemic inflammation. The blood pressure of these animals are all high (due to the strain of the rats themselves), but there was no significant difference between the non-inflamed and arthritis-induced animals.

 

The key findings in our model is that animals that have mono-arthritis and joint degradation show an increase in the level of the inflammatory mediator TNF alpha (and likely other cytokines such as IL-17). More importantly, we found that these rats develop hemorrhagic stroke in the brain when they are at an equivalent human age of 30-40 years old, suggesting that hypertension and chronic inflammation synergistically promote the induction of hemorrhagic stroke. There is an associated increase in brain inflammation and brain damage (Figure 2; in press Randell et al., PeerJ).  Moreover, the study investigated the potential interaction of dietary salt (with 4% NaCl) and chronic inflammation induced by mono-arthritis, which showed a resulting increase in elevation of systemic inflammatory mediator TNF alpha and the elevated incidence of fatal stroke. The length and timing that the animals are placed on the 4% high salt diet mimics an individual who is on a typical North American diet (relatively high in salt, ~5.8-11.8 g/day) from approximately 15 years of age into their middle age years. We have found compelling evidence that the incidence and severity of hemorrhagic stroke and the degree of brain inflammation and damage are dramatically higher in our arthritic-hypertensive rats when they are put on a specific high salt diet of 4% NaCl from 5 weeks of age (see article and Figure 3).

 

We believed that the brain vessels (MCAs) observed in our hypertensive mono-arthritic animal models undergo very similar changes as the brain vessels of stroke patients or other stroke-prone animal models (Randell et al, PeerJ 2016), but with the key difference that it is the induction of arthritis and the systemic inflammation (akin to what patients with autoimmune disease experience), which predispose the animals to fatal stroke. Also, activation of inflammatory signaling is likely causing a decrease in theMCAs ability to respond to pressure dependent constriction by affecting TRP channels and endothelial and smooth muscle functions directly. Our model enables us to investigate the interaction between the inflammatory mediators, the activation of inflammatory receptors, changes to the contractile proteins, and the involvement of high salt further. I believe that this information is very important in order to develop appropriate and effective management strategies for the aging population suffering from RA. To this end, our hypertensive-arthritic model is ideal for investigating how salt consumption impacts the severity of a chronic inflammatory disease and its propensity to potentiate fatal stroke.

The goal of our research is to define how we arrive at fatal stroke in presence of a systemic inflammatory injury, the type of damage the arthritis causes systemically and centrally, and to offer appropriate treatment options that are patient specific to the arthritic population to minimize fatal stroke.

 

 

fig1

Figure 1 describes the mechanism by which we have created the animal model. The SHR strains were bred inhouse and weaned at 5 weeks. They were divided to those who received High Salt Diet (HS; 4% salt) or regular chow diet (RD; 0.58% salt). At around 20 weeks of age, they received 0.07ml injection intradermally on the left foot pad either CFA or saline and monitored for 21 days. On the day of sacrifice, the brains were collected after Evans blue dye infusion to determine hemorrhage formation, MCAs were collected for functional analysis and brains were fixed and stained for histological analysis.

 

fig2

Figure 2: Immunofluorescence and H&E stains of the brains of hypertensive mono-arthritic and control (CFA and Sal) animals showing microglia activation and astrocyte spread from the cortex to the neocortex , and neural damage incurred by CFA.

 

fig3

Figure 3: Immunofluorescence and H&E stains of the brains of hypertensive mono-arthritic and control (CFA and Sal) animals placed on 4% salt diet showing microglia activation and astrocyte spread from the cortex to the neocortex , and neural damage incurred by CFA. Having the animals on the high salt diet itself caused an increase in microglia activation , astrocyte spread and increase in nuclear vacuolation (indicative of neural damage). Induction of systemic inflammation (CFA) exascerbated the brain damage and central inflammation.

 

Acknowledgements:

This work was supported by RDC/Ignite Fund awarded to Noriko Daneshtalab.

 

fig4Contact:

Dr. Noriko Daneshtalab, PhD

Assistant Professor, School of Pharmacy, Cross Appointment to Faculty of Medicine Memorial University of Newfoundland

Health Sciences Centre 300 Prince Philip Drive St. John’s, NL  |   A1B 3V6

Email: norikod@mun.ca

Website: http://www.mun.ca/pharmacy/research/daneshtalab.php T  709 777 2218   |   F  709 777 7044

 

 

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