Efficacy Assessment of Cerebral Perfusion Augmentation through Functional Connectivity in an Acute Canine Stroke Model
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* Chisondi S. Warioba
* Mira Liu
* Sagada Peñano
* Timothy J. Carroll
* Sean Foxley
* Gregory Christoforidis

## Abstract

**BACKGROUND AND PURPOSE:** Ischemic stroke disrupts functional connectivity within the brain's resting-state networks (RSNs), impacting recovery. This study evaluates the effects of norepinephrine and hydralazine (NEH), a cerebral perfusion augmentation therapy, on RSN integrity in a hyperacute canine stroke model.

**MATERIALS AND METHODS:** Fifteen adult purpose-bred mongrel canines, divided into treatment and control (natural history) groups, underwent endovascular induction of acute middle cerebral artery occlusion (MCAO). Postocclusion, the treatment group received intra-arterial norepinephrine (0.1–1.52 µg/kg/min, adjusted for 25–45 mm Hg above baseline mean arterial pressure) and hydralazine (20 mg). Resting-state fMRI (rs-fMRI) data were acquired with a 3T scanner by using a blood oxygen level dependent-EPI sequence (TR/TE = 1400 ms/20 ms, 2.5 mm slices, 300 temporal positions). Preprocessing included motion correction, spatial smoothing (2.5 mm full width at half maximum), and high-pass filtering (0.01 Hz cutoff). Functional connectivity within RSNs were analyzed through group-level independent component analysis and weighted whole-brain ROI-to-ROI connectome, pre- and post-MCAO.

**RESULTS:** NEH therapy significantly maintained connectivity post-MCAO in the higher-order visual and parietal RSNs, as evidenced by thresholded statistical mapping (threshold-free cluster enhancement *P*corr > .95). However, this preservation was network-dependent, with no significant (*P*corr < .95) changes in the primary visual and sensorimotor networks.

**CONCLUSIONS:** NEH demonstrates potential as a proof-of-concept therapy for maintaining RSN functional connectivity after ischemic stroke, emphasizing the therapeutic promise of perfusion augmentation. These insights reinforce the role of functional connectivity as a measurable end point for stroke intervention efficacy, suggesting clinical translatability for patients with insufficient collateral circulation.

## ABBREVIATIONS:

FWE
:   family-wise error
ICA
:   independent component analysis
MCAO
:   middle cerebral artery occlusion
NEH
:   norepinephrine and hydralazine
rs-fMRI
:   resting-state fMRI
RSN
:   resting-state network
TFCE
:   threshold-free cluster enhancement

SUMMARY SECTION

#### PREVIOUS LITERATURE:

Current understanding emphasizes the vulnerability of resting-state networks (RSNs) to ischemic stroke and the potential of perfusion augmentation strategies to influence poststroke recovery. The efficacy of such treatments in preserving functional connectivity within RSNs remains a key area of investigation.

#### KEY FINDINGS:

Norepinephrine and hydralazine (NEH) therapy preserved functional connectivity in higher-order visual and parietal networks poststroke in canines, with the treatment effect proving network-specific.

#### KNOWLEDGE ADVANCEMENT:

This study advances the concept of targeted perfusion augmentation, demonstrating NEH's role in neural network protection and its potential for clinical stroke intervention.

Ischemic stroke, a leading cause of mortality and chronic disability worldwide, imposes a serious challenge for both acute management and long-term rehabilitation.1,2 The intricate pathophysiology of stroke, primarily the abrupt disruption of cerebral blood flow leading to neuronal death, necessitates advancements in therapeutic strategies beyond the acute phase, aiming to restore and enhance neurologic function and quality of life.3

Recent strides in neuroimaging, particularly resting-state fMRI (rs-fMRI), have opened new avenues in understanding the brain’s intrinsic functional connectivity.2⇓-4 This noninvasive technique provides insights into the spontaneous neural activity across various resting-state networks (RSNs), which are critical for various cognitive and motor functions.4⇓⇓⇓-8 The dynamic changes in these networks poststroke are increasingly recognized for their potential implications in predicting recovery trajectories and guiding rehabilitation efforts.4⇓⇓⇓⇓⇓⇓⇓-12

Building upon this premise, our study introduces a therapeutic strategy aimed at augmenting cerebral perfusion in the acute phase of ischemic stroke. Utilizing a combination of norepinephrine and hydralazine (NEH) administered intra-arterially, we hypothesize that this intervention can modulate the functional connectivity within key RSNs, thereby preserving or potentially enhancing neurologic function. This rationale is rooted in the dual mechanism of NEH, namely elevating systemic blood pressure to improve perfusion and directly inducing vasodilation to augment blood flow to ischemic regions, including the penumbra, which is critically vulnerable in the aftermath of a stroke.

This investigation uses a canine model of hyperacute ischemic stroke, induced via middle cerebral artery occlusion (MCAO), to examine the effects of NEH therapy on functional connectivity across identified RSNs through rs-fMRI.13 Our study is poised to contribute significantly to the understanding of cerebral perfusion augmentation as a therapeutic mechanism, potentially setting the stage for translational applications in stroke recovery and rehabilitation strategies.

In focusing on the alterations in RSNs, we aim not only to highlight the neuroprotective potential of NEH therapy but more importantly to underscore the relevance of functional connectivity as a critical end point in evaluating the efficacy of stroke interventions. Through this research, we aspire to enrich the dialogue within the neuroscientific community regarding the integration of advanced imaging techniques and pharmacologic innovations in crafting the next generation of stroke therapies.

## MATERIALS AND METHODS

### Experimental Design and Canine Model

Fifteen adult purpose-bred mongrel canines (average weight 25 ± 5 kg, comprising 12 females and 3 males, with an average age of 4.2 ± 3.3 years) were selected to investigate the neuroprotective effects of cerebral perfusion augmentation via NEH on rs-fMRI-determined functional connectivity. The 15 animals were split into a control and treatment group, with 7 per group due to the death of a subject. The study spanned 2 days, starting with baseline MRI scans under general anesthesia, which was meticulously maintained by using a combination of propofol (25 µg/kg/min) for systemic sedation, rocuronium bromide (0.4–0.6 mg/kg) for muscle relaxation, and isoflurane (1% end-tidal concentration) to ensure stable anesthesia depth without compromising cerebral perfusion.14 The anesthesia protocol was the exact same for both groups.

Blood analysis, including pH, PaO2, PaCO2, glucose, hematocrit, and hemoglobin levels, was conducted after intra-arterial access and immediately postmicrosphere injection. Mean arterial blood pressure was continuously monitored and maintained at 80–100 mm Hg by using a 5F intra-arterial sheath. Intravenous neosynephrine was administered as necessary to manage mean arterial pressure. Central venous pressure was tracked with a central venous catheter, while intracranial pressure monitoring was used during the acute phase of experimental MCAO. Capnographic end-tidal carbon dioxide levels were maintained at 35–40 mm Hg, verified periodically through direct arterial sampling for PaCO2 accuracy. Body temperature was monitored by a rectal probe and regulated by using heating pads, and blood glucose levels were kept below 120 with IV insulin when needed.

#### Induction of Ischemic Stroke.

On the second day, canines underwent endovascular MCAO at the M1 segment. This procedure was achieved by using embolic occlusion coils at the M1 and carotid terminus, aimed at simulating a hyper-acute ischemic stroke. Two and one-half hours postocclusion, detailed rs-fMRI scans were conducted to assess the impact on brain functional connectivity. Occlusions were randomized between the left and right MCA; however, for imaging analyses, all images were flipped so the occluded side was the right hemisphere, as is common practice in lesion studies.15,16

#### Therapeutic Intervention.

Before the administration of any therapeutic intervention, successful MCAO was confirmed through arteriographic imaging, highlighting the cessation of blood flow in the M1 segment of the MCA. This step ensured the accurate emulation of an ischemic stroke scenario. Pial collateral recruitment was then assessed through contrast-enhanced arteriography. After this, 7 canines in the treatment group received a precise therapeutic regimen, consisting of an intra-arterial infusion of norepinephrine (0.1–1.52 µg/kg/min) to elevate mean arterial pressure by 25–45 mm Hg above baseline, alongside a single 20 mg dose of hydralazine. This treatment, initiated 30 minutes postocclusion, aimed to enhance cerebral perfusion through a combined approach of pressure elevation and vasodilation.

In contrast, the control group underwent standard care, maintaining mean arterial pressure within the physiologic range (80–105 mm Hg) to serve as a baseline. This controlled approach allowed for the evaluation of the treatment impact of NEH on function connectivity and cerebral perfusion.

Pial collateral scores were determined by a board-certified neuroradiologist based on the extent and timing of collateral blood flow in the ischemic territory, by using a scoring system where scores greater than 8 indicated robust collateral circulation (“good”), and scores less than or equal to 8 signified inadequate collateral flow (“poor”). This scoring was critical for stratifying the subjects based on their innate cerebral vascular response to ischemia.

#### rs-fMRI Protocol and Analysis.

rs-fMRI scans were acquired on a 3T MRI scanner (Achieva, Philips), tailored to the specific requirements of rs-fMRI to capture the nuances of brain functional connectivity. The imaging protocol utilized a blood oxygen level dependent-sensitive EPI sequence with TR/TE =1400 ms/20 ms, voxel size of 2.5 mm, and 300 temporal positions.

Before the acquisition, canines were positioned within the scanner to maximize comfort and minimize movement, with padding and restraints used as necessary. The rs-fMRI data preprocessing involved several steps to prepare the data for analysis: motion correction by using FSL MCFLIRT tool (version v6.01; [http://fsl.fmrib.ox.ac.uk/fsl/fslwiki/MCFLIRT](http://fsl.fmrib.ox.ac.uk/fsl/fslwiki/MCFLIRT)),17⇓⇓⇓⇓-22 spatial smoothing with a Gaussian kernel of 2.5 mm full width at half maximum to improve the signal-to-noise ratio, and temporal filtering with a high-pass filter (0.01–0.1 Hz) to reduce low-frequency drift and high-frequency physiologic noise. Subsequent data analysis used group-level independent component analysis (ICA) with the FSL MELODIC tool ([http://fsl.fmrib.ox.ac.uk/fsl/fslwiki/MELODIC](http://fsl.fmrib.ox.ac.uk/fsl/fslwiki/MELODIC)) to identify and separate out the different RSNs, including the primary visual, sensorimotor, higher-order visual, and parietal networks. Each RSN was then individually assessed for changes in functional connectivity before and after the therapeutic intervention.23 Additionally, whole-brain weighted ROI-to-ROI connectivity analysis was conducted with 262 cortical-only ROIs based on a 2020 canine atlas by Johnson et al24 (this same atlas was used for co-registration of rs-fMRI data). Circular connectivity plots (connectograms) were created based on this analysis to adequately visualize pre- versus postocclusion connectivity differences (Fig 1). Connectograms were created in CONN (Functional connectivity toolbox) in Matlab (MathWorks; version R2024a).25

![FIG 1.](http://www.ajnr.org/https://ajnr-sso.highwirestaging.com/content/ajnr/45/9/1214/F1.medium.gif)

[FIG 1.](http://www.ajnr.org/content/45/9/1214/F1)

FIG 1. 
Comparative analysis of functional connectivity and perfusion in a canine model poststroke. *A*, The functional connectivity network in the control group after occlusion, with the thickness and color of the lines indicating the strength of connectivity between regions (atlas-based; Johnson et al24). *B*, Demonstrates the corresponding loss of perfusion (qCBF measured in mL/100 g/min) in the control group, with cooler colors representing reduced blood flow and cell death. *C*, Displays the functional connectivity network in the NEH-treated group postocclusion, also reflecting connectivity strength via line color and thickness. *D*, Shows the NEH group’s perfusion, where warmer colors indicate preserved or augmented blood flow relative to the control and the cooler colors in the brain in this case are above the threshold for cell death. *E*, Depicts the control group’s preocclusion baseline functional connectivity for comparison. *B* and *D*, Quantitative perfusion images (color bar ranging from 0–170 mL/100 g/min) that are included (with author permission) for purely demonstrative purposes from an external quantitative perfusion study (Liu et al10) to visually correlate the changes in functional connectivity with alterations in perfusion, highlighting the protective effects of NEH on maintaining perfusion after stroke in a single subject.

#### Statistical Approach.

To quantitatively assess the impact of NEH treatment on the functional connectivity within the identified RSNs, a comprehensive statistical analysis was conducted. Initially, individual component maps generated by ICA were compared between pre- and postintervention scans across both treatment and control groups. This comparison utilized threshold-free cluster enhancement (TFCE); a technique designed to robustly handle the issue of multiple comparisons across spatially extended statistical maps without the need for arbitrary thresholding.

Family-wise error (FWE) correction was applied to adjust for the multiple testing inherent in neuroimaging analyses, ensuring that reported changes in connectivity were not due to chance. Statistical significance was set at a *P* value of < .05 after FWE correction. Additionally, a linear mixed-effects model was implemented to account for within-subject variations over time and between the different RSNs. This model included terms for treatment group, time point (pre- versus postintervention), and their interaction, allowing for the examination of how NEH treatment specifically influenced the dynamics of RSN connectivity post-MCAO.

All statistical analyses (paired *t* tests) were performed by using FSL Randomize ([http://fsl.fmrib.ox.ac.uk/fsl/fslwiki/Randomise](http://fsl.fmrib.ox.ac.uk/fsl/fslwiki/Randomise)), providing a robust platform for implementing the complex models required to interpret the rs-fMRI data accurately. The results from these analyses not only elucidated the direct effects of NEH on brain network connectivity but also provided insights into the broader implications of perfusion augmentation therapy for restoring functional connectivity following acute ischemic stroke. All scripts are available upon request.

#### Ethical Considerations.

All experimental procedures received approval from the Institutional Animal Care and Use Committee at The University of Chicago, which is an AAALAC International–accredited facility. The study was designed and conducted in strict accordance with the National Institutes of Health guidelines for the humane care and use of laboratory animals, ensuring ethical and responsible research practices.

## RESULTS

Utilizing a whole-brain cortical atlas-based ROI-to-ROI analysis, significant (*P*corr > .95) global declines in functional connectivity were observed in the control group after occlusion. These changes were captured in the connectograms of Fig 1, depicting a generalized weakening of interregional connectivity.

Within specific RSNs, our analysis revealed a statistically significant (*P*corr > .95) decrease in functional connectivity in the higher-order visual network (RSN3) postocclusion in the control group. Conversely, in the NEH-treated group, the parietal network (RSN4) showed a significant increase in functional connectivity after treatment compared with the preocclusion state. This contrast suggests that NEH treatment may confer a protective or restorative effect on the functional integrity of RSNs after stroke (Fig 2). All RSNs identified, including those without statistically significant changes, were anatomically verified by a canine neuroimaging expert, ensuring the reliability of functional connectivity mapping.

![FIG 2.](http://www.ajnr.org/https://ajnr-sso.highwirestaging.com/content/ajnr/45/9/1214/F2.medium.gif)

[FIG 2.](http://www.ajnr.org/content/45/9/1214/F2)

FIG 2. 
RSN connectivity comparisons pre- and postocclusion in control and NEH-treated canines. *Top row*: Baseline functional connectivity within 4 identified RSNs in a canine brain model. From left to right, RSN1 (primary visual), RSN2 (sensorimotor), RSN3 (higher-order visual), and RSN4 (parietal) are displayed, with the intensity of activation indicated by *Z* score values, where warmer colors represent stronger connectivity. *Bottom row*: Significant contrasts derived from a paired *t* test by using FSL Randomize, indicating changes in functional connectivity within the control and NEH-treated groups. Bottom left (RSN3 contrast: Control preocclusion versus Control postocclusion) demonstrates a reduction in connectivity postocclusion within the control group, as evidenced by warmer colors in the preocclusion state. Bottom right (RSN4 contrast: NEH postocclusion versus NEH preocclusion) shows enhanced connectivity postocclusion in the NEH-treated group compared with preocclusion, suggesting an improvement in connectivity within the parietal network. These contrasts have been thresholded for significance at *P*corr > .95 by using TFCE, highlighting the impact of NEH treatment on RSN connectivity after induced occlusion. All RSNs and statistical maps are overlayed on a canine template atlas (Johnson et al24).

Statistical substantiation of these findings was provided by paired *t* tests and TFCE with FWE correction. The meaningful changes in RSN3 and RSN4 surpassed the threshold of TFCE *P*corr > .95, underscoring the significant impact of occlusion and NEH treatment on these networks. The absence of such significant findings in the primary visual (RSN1) and sensorimotor (RSN2) networks indicates that the effects of stroke and the neuroprotective influence of NEH may be network-specific.

## DISCUSSION

The present study explored the therapeutic impact of cerebral perfusion augmentation via NEH on functional connectivity alterations in RSNs after hyperacute ischemic stroke in canines. Consistent with our hypothesis, NEH treatment preserved functional connectivity within the parietal network (RSN4) postocclusion and, notably, in the higher-order visual network (RSN3).23 This enhancement of connectivity suggests a potential neuroprotective effect, possibly mitigating the typical functional deterioration seen in these networks poststroke.

Interestingly, while NEH demonstrated beneficial effects in certain RSNs, others like the primary visual (RSN1) and sensorimotor (RSN2) did not exhibit significant changes. This differential response underscores the complexity of cerebral autoregulation poststroke and highlights the necessity of targeted therapeutic approaches. It further aligns with current perspectives on the heterogeneity of stroke impacts across different neural circuits, suggesting that therapeutic efficacy may be network-specific, resonating with findings from prior studies on stroke and connectivity.4,15,16,25⇓⇓⇓⇓⇓-31

Our study suggests that NEH may augment perfusion effectively in specific brain regions, thus preserving or enhancing their functional connectivity. This could have profound implications for stroke recovery, as it supports the premise that pharmacologically maintained perfusion in the acute phase can improve outcomes by preserving network integrity.

However, the intricacies of this relationship must be navigated with caution. While RSN3 and RSN4 showed significant connectivity improvements, suggesting a fortification of network resilience, this effect was not universal across all networks. This finding may be reflective of a complex interplay between the pharmacodynamics of NEH, the inherent resilience of different networks, and the degree of ischemic insult. It compels a deeper investigation into the nuanced ways different RSNs respond to ischemic conditions and the potential for tailored pharmacological intervention.

Considering the limitations, while our canine model offers a translational platform, differences in human and canine cerebrovascular dynamics warrant careful extrapolation of these findings. The sample size, though yielding notable results, constrains the broad applicability of the findings. Future studies with larger cohorts and diverse stroke models could address this limitation and confirm the generalizability of the results.

Implications for patient care are cautiously optimistic. The identification of functional connectivity as a quantifiable measure of therapeutic efficacy could inform the management of stroke patients, potentially leading to individualized treatment protocols. Nonetheless, the translation from canine models to clinical practice must navigate the complexities of human stroke pathology and the variability in patient responses to treatment.

For future research, several trajectories appear promising. Investigating the temporal dynamics of NEH therapy, examining dose-response relationships, and extending the analysis to include a broader range of RSNs could provide deeper insights. Studies might also explore the potential of NEH in combination with established reperfusion therapies like thrombolysis.

## CONCLUSIONS

Our findings underscore the role of NEH as a proof of concept for cerebral perfusion augmentation therapies in acute ischemic stroke, showing significant preservation of functional connectivity in key RSNs, notably in higher-order visual and parietal networks, within a canine model. This indicates that targeted perfusion augmentation is critical for network integrity preservation. The variability in network response underscores the necessity for tailored therapeutic approaches. These results, alongside the demonstrated utility of functional connectivity as a therapy efficacy biomarker, lay the groundwork for integrating perfusion augmentation strategies into stroke management, particularly benefiting patients with limited collateral circulation. Moving forward, the application of NEH in the acute stroke phase opens promising avenues for neuroprotection, meriting further research for clinical translation.

## Acknowledgments

We thank M. Liu et al for data from their 2022 publication that allowed for perfusion comparisons to the functional data.

## Footnotes

*   The authors acknowledge the following grants from the National Institutes of Health (NIH) and National Science Foundation (NSF) for making this work possible: NIH R01NS093908, NIH 5R25GM109439-09, and NSF GRFP 2140001 and 1746045.

*   [Disclosure forms](https://www.ajnr.org/sites/default/files/additional-assets/Disclosures/September%202024/0140.pdf) provided by the authors are available with the full text and PDF of this article at [www.ajnr.org](http://www.ajnr.org).

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*   Received February 7, 2024.
*   Accepted after revision April 16, 2024.


*   © 2024 by American Journal of Neuroradiology