*p*-value by non-parametric Mann–Whitney test. All values are median (with interquartile range –IQR unless stated otherwise).

#### *3.3. Coagulation Outcomes*

The clotting time became prolonged (*p* = 0.008) and MCF was weakened in both InTEM (*p* = 0.001) and ExTEM (*p* = 0.001) in the study dogs after hemorrhage. In addition to having a better clot firmness (or MCF) prior to hemorrhage, dogs treated with resveratrol also had a higher peak thrombin generation potential at 1-h after hemorrhage (Table 3). MCF on the InTEM appeared to remain better in the resveratrol group during the period of hemorrhage after adjusting for volume of blood withdrawn in each animal in the linear mixed model analysis (Table 6).

**Table 6.** Linear mixed models showing the associations between resveratrol treatment with maximum clot firmness on the Rotational Thromboelastometry (ROTEM®) of the anesthetized greyhound dogs before and after induced hemorrhage (*n* = 12) after adjusting for volume of blood removed using unstructured covariance structure. MCF = maximum clot firmness.


#### *3.4. Renal Histopathology*

Notably, severe tubular injury (evidence of ischemic coagulative necrosis) was not identified in any specimen. In the 20 randomly selected 200× fields of cortex (due to the lack of lesions in the deeper regions of the renal parenchyma) for each kidney examined, there was evidence of injury (loss of brush border, singly dead cells, sloughed cellular debris) in the study animals. Both control and resveratrol treated dogs had normal to mildly injured tubules (Figure 3A,B). One resveratrol-treated dog had evidence of moderate tubular injury (Figure 3C).

**Figure 3.** (**A**) Photomicrograph from a control dog with mild acute tubular epithelial injury, characterized by loss of the brush border and simplification of the tubular epithelium. There are 2 sloughed necrotic cells within the lumen. (**B**) Photomicrograph from a treated dog with mild acute tubular epithelial injury characterized by the presence of many sloughed epithelial cells within a tubular lumen. (**C**) Photomicrograph from a treated dog that had moderate acute tubular epithelial injury, characterized by simplification and attenuation of tubular epithelium (asterisk) that was more frequently observed than in dogs with mild injury. Periodic acid Schiff, 40 × magnification.

#### *3.5. Renal Transmission Electron Microscopy (TEM)*

Samples from two control dogs and two treated dogs were further evaluated using TEM. All four dogs had evidence of loss of the apical brush border, cellular swelling and intraluminal cellular fragments (Figure 4). *Proximal tubules* were most frequently damaged but distal convoluted tubules were occasionally affected. Ultra-structurally, there were no obvious differences between the two dogs in each study group (although this was not formally tested statistically because of the small sample size of this sub-group). Dogs that had minimal injury in the histology specimen had similar ultrastructural lesions on the TEM as dogs that had mild and moderate histologic lesions.

**Figure 4.** (**A**) Transmission electron micrograph from a control dog that had minimal acute tubular epithelial injury in the histology sample. The sample submitted for TEM showed sloughed cellular debris (circled) and electron dense cytoplasmic material (mineralization) (asterisk). There is denuded tubular basement membrane. Bar is 2 micron. (**B**) Transmission electron micrograph from a treated dog that had mild acute tubular epithelial injury in the histology sample. There is loss of the apical brush border of multiple cells (circled) whereas neighboring cells maintain intact brush borders. Bar is 20 micron. (**C**) Transmission electron micrograph from a control dog that had minimal injury in the histology sample. There is loss of the apical brush border (circled) as well as sloughed cells within the tubular lumen (asterisk). Bar is 2 micron.

#### **4. Discussion**

This study showed that seven days of oral resveratrol treatment in greyhound dogs improved their blood pressure tolerance to induced hemorrhage; that is, treated animals required a larger amount of blood loss to develop the same degree of hypotension. There was also a signal to suggest that resveratrol might improve clot strength and thrombin generation. The potential renal protective effect of resveratrol was, however, not observed both in terms of biochemical and histological assessments. These findings are clinically relevant and require further discussion.

First, a blood pressure targeted hemorrhage model was used in this study in an attempt to assess whether resveratrol could improve the maintenance of blood pressure during hemorrhage. Whether this was a better model than a model that induces a fixed amount of blood loss remains uncertain [28]. The volume of blood loss to achieve the predetermined MAP in our control animals (median 54.9, range 42.5–58.9 mL/kg) was consistent with the results of another study (53 mL/kg, 95% confidence interval 48–57) [29]. As such, the greater amount of blood loss needed to achieve and maintain the same degree of hypotension (and OER in the central venous blood) in the resveratrol-treated dogs (median 63.8, range 57.8–78.6 mL/kg) suggests that oral resveratrol pretreatment may have genuinely assisted the dogs in maintaining a better blood pressure during acute bleeding. This beneficial effect of resveratrol may theoretically, at least in part, be related to activation of SIRT1 gene, its estrogen agonist effect, or interactions with other targets or receptors that are relevant to pathogenic process of I-R injury [6–8]. Because we could not detect resveratrol levels in most blood samples of our resveratrol-treated dogs, the precise mechanisms how resveratrol can help to maintain the blood pressure remains unclear, but modification of gut microbiota is one possibility [30]. Nonetheless, the dogs in the resveratrol group did have a lower MAP and pH in the arterial and venous blood at the end of 60-min of hemorrhage. Repeating the study with a larger sample size, using a volume-guided hemorrhagic model using preset volume of blood loss, and extending the study duration to include survival time and effect on gut microbiota would be helpful to confirm the benefits of resveratrol, and clarify the mechanisms through which oral resveratrol pretreatment improves cardiovascular tolerance to hemorrhage.

Second, all study dogs had an increase in many serum and urinary AKI biomarkers induced hemorrhage; and these changes were associated with histopathological evidence of AKI. This was not surprising as a reduction in blood volume and pressure would certainly compromise renal blood flow, triggering a reduction in glomerular filtration rate and induction of renal ischemia [31]. As such, the hemorrhagic model used in our study was sensitive enough to induce bleeding-related AKI [32]. Despite this, there were no significant difference in all the renal biomarkers and histological changes between the resveratrol and control groups. This negative result could be explained by a number of reasons. First of all, a much lower dose (per body weight) of resveratrol was used in this study and outcomes were assessed in a shorter time frame than in the rat models in previous studies [6–8,16,27,33]. Furthermore, a small sample size could undermine the statistical power of this study. Of the dogs that were treated with resveratrol, 40% (2/6 dogs) developed stage-1 AKI (defined by an increment in SCr > 26.4 µmol/L) compared to 100% of the dogs in the control group, even though this difference did not reach statistical significance. Another possibility is that resveratrol might have different renal effects in different animal species. To the best of our knowledge, using resveratrol to prevent AKI during hemorrhage in dogs (in contrast to rats [16]) has not been assessed previously. Some types of intravenous fluid used for resuscitation can also have an adverse effect on the kidneys. Although the same types and similar quantities of intravenous fluid (Compound Sodium Lactate for maintenance at 10 mL/kg/hour and Gelofusine® to restore normal blood pressure: see Table 4) were used for both groups in this study, it is possible that resveratrol's renal protective effect—if there is one—was confounded or overwhelmed by a potential harmful or beneficial effect of Gelofusine® on the kidneys. Previous studies have shown that using Gelofusine® for resuscitation was less likely to induce AKI compared to the older forms of intravenous starches in humans [34], but recent animal studies have shown that Gelofusine® may induce AKI compared to crystalloids or fresh whole blood [35]. As such, it would be better to use crystalloid fluid alone during the reperfusion period in similar experiments in the future.

An interesting and important finding of this study was the positive association between resveratrol, clot firmness and thrombin generation. A previous study has showed that resveratrol could help to preserve platelet function in stored blood [36]. Conversely, resveratrol has also been shown to induce platelet dysfunction and coagulation derangements in a few other experimental studies [19,20,37,38]. Given hemostasis is a paramount outcome in any hemorrhagic situations, the effect of resveratrol on bleeding tendency requires further investigation.

Finally, we need to acknowledge the limitations of this study. The circumstances of how our study animals were recruited had limited our ability to address the important issue of survival time after hemorrhage. It is important to note that race-trained greyhounds are highly adapted to anaerobic tissue metabolism during exercise, modulating their cardiovascular, hemostatic and homeostatic responses to hemorrhage [39,40]. Prolonged anesthesia (5 h in this study) might have interfered with the normal cardiovascular compensatory responses to bleeding. In addition, pure µ-agonist opioids, such as the fentanyl used in this study, are known to cause venous and arterial blood wall relaxation, via endothelium and opioid-receptor independent mechanisms in various species [41,42]. Similarly, isoflurane also has a dose-dependent vasodilatory and negative inotropic effect, rendering our study animals more likely to develop hypotension after hemorrhage [43,44]. Nonetheless, a parallel-arm randomized-controlled design should have balanced the confounding effects of these factors.

#### **5. Conclusions**

This exploratory study showed that seven days of oral resveratrol treatment prior to bleeding improved greyhound dogs' blood pressure stability in response to severe hemorrhage and possibly also coagulation profiles compared to no resveratrol treatment. A renal protective effect of resveratrol in hemorrhage was, however, not observed. An adequately-powered study using a volume-targeted hemorrhage model assessing the benefits of resveratrol, including survival time, is needed before this dietary supplement can be recommended prior to exposure to hemorrhage.

**Author Contributions:** Conceptualization, A.L.R. and K.M.H.; Methodology, J.D., A.L.R. and K.M.H.; Investigation, J.D., A.L.R., C.R.S., R.E.C. and S.C.W.; Formal Analysis, K.M.H.; Resources, J.D., A.L.R., C.R.S., R.E.C., S.C.W. and K.M.H.; Writing—Original Draft, J.D. and A.L.R.; Writing—Review and Editing, J.D., A.L.R., C.R.S., R.E.C., S.C.W. and K.M.H.; Visualization, J.D., R.E.C. and K.M.H.; Supervision, A.L.R. and K.M.-H.; Project Administration, J.D. and A.L.R.; Funding Acquisition, K.M.-H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Australian Society of Anaesthetists Jackson Rees Research Award in 2015.

**Institutional Review Board Statement:** This study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Animal Ethics Committee of Murdoch University (R2726/15).

**Informed Consent Statement:** Written informed consent was obtained from the animal owners to publish this paper.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The authors thank Alan Flechtner and Anne Saulsbery from the Comparative Mouse Phenotyping Shared Resource (Cancer Center Support Grant P30 CA016058) for histopathology and electron microscopy specimen preparation, and Jeffrey Lipman from the University of Queensland Centre for Clinical Research for measurement of plasma resveratrol concentration.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

## **References**

