Inhibition of iNOS reduced cerebral vasoconstriction from chronic cocaine and enhanced angiogenesis in the prefrontal cortex

Abstract

Cocaine is a highly addictive drug associated with neurovascular complications, such as transient ischemic attacks (TIAs) and strokes. Neuroimaging studies have documented marked decreases in cerebral blood flow (CBF) in the brain of cocaine abusers. Recently we showed astrocytes’ involvement in cocaine-induced vasoconstriction and associated cortical ischemia in the mouse brain. We hypothesize that inducible nitric oxide synthase (iNOS) regulation plays an important role. To investigate this, we applied ultra-high resolution optical coherent tomography (μOCT) to capture 3D vascular images of the prefrontal cortex (PFC) in mice before and after chronic cocaine treatment. We compared vascular parameters including vessel diameter and microvascular density. Our results revealed that chronic cocaine exposure led to marked vasoconstriction in wild-type (WT) mice. In contrast, these effects were significantly attenuated in iNOS knockout (iNOS-KO) mice and in WT mice pretreated with the iNOS inhibitor L-NMMA (iNOS-Inh), thus indicating iNOS’s involvement in the vascular changes triggered by cocaine. Furthermore, both iNOS-KO and iNOS-Inh mice exhibited increased microvascular density following cocaine exposure. This suggests a compensatory angiogenic response to mitigate for the vasoconstriction. Complementing structural imaging, we utilized a multimodal imaging platform (MIP) to assess hemodynamic responses during cocaine or saline administration. Consistent with our μOCT findings, WT mice displayed significant reductions in CBF and oxyhemoglobin levels following cocaine treatment. These hemodynamic impairments were notably diminished in iNOS-KO mice, supporting the role of iNOS in mediating cocaine-induced cerebrovascular dysfunction. Together, these findings suggest that iNOS contribute significantly to cocaine-induced vasoconstriction and cerebral hypoperfusion. Thus, the inhibition of iNOS could be a potential therapeutic strategy to reduce the risk of cerebral ischemia in individuals with cocaine use disorder.

1. Introduction

Cocaine is a highly addictive drug associated with a range of neurovascular complications, including transient ischemic attacks (TIAs) and strokes (Treadwell and Robinson, 2007). While the exact mechanisms underlying cocaine-related cerebrovascular impairments are not fully understood, it is believed that cocaine-induced vasoconstriction plays a significant role (Toossi et al., 2010). Indeed, numerous clinical brain imaging studies have reported widespread reductions in cerebral blood flow (CBF) and volume in individuals with cocaine use disorder (Volkow et al., 1988; Luo et al., 2009), most markedly in the prefrontal cortex (PFC) and in rodents chronically expose to cocaine (Du et al., 2020a; Zhang et al., 1996; Chen et al., 2016a).

Recent rodent studies have demonstrated that astrocytes contribute to cocaine-induced vasoconstriction and ischemic responses (Hayashi et al., 2003; Krupinski et al., 1994a), which we hypothesize may involve inducible Nitric Oxide Synthase (iNOS) involvement. Nitric oxide (NO), produced by iNOS in astrocytes, whose signaling pathway is activated under conditions of reduced CBF and hypoxia (Beck and Plate, 2009; Mo et al., 1998). However, the role of iNOS in mediating the vascular effects of cocaine has not been thoroughly investigated. In this study, we hypothesized that iNOS is involved in the vascular changes associated with cocaine (Alvarez et al., 2008). We specifically hypothesized that inhibition of iNOS expression would attenuate cocaine-induced vasoconstriction and cerebral ischemia.

To test these hypotheses, we used optical imaging techniques to evaluate the impact of chronic cocaine on vascular diameter and density and track hemodynamics in the PFC in vivo, both in wild type (WT) and iNOS knock out (iNOS-KO) mice. Specifically, the ultra-high resolution optical coherent tomography (μOCT) was used to capture 3D microvasculature and CBF images in the PFC. This allowed us to quantitatively assess vessels diameters and microvascular density before and after chronic cocaine. In parallel, we used a lab-developed multimodal imaging platform (MIP) to track dynamic brain responses to cocaine periodically during chronic cocaine exposures. Through a cranial window, we monitored various parameters, including changes in total hemoglobin (blood volume, Δ[HbT]), oxygenated hemoglobin (Δ[HbO₂]), and deoxygenated hemoglobin (Δ[HbR]). This platform enabled longitudinal assessment of the hemodynamic response to acute cocaine (1 mg/kg, i.v.) on Day 1, ~Day 7, and Day 14 during a two-week period of daily intraperitoneal (i.p.) cocaine treatment (30 mg/kg/day). Four experimental groups were studied: 1) WT mice treated with saline as controls; 2) WT mice treated with cocaine; 3) iNOS-KO mice treated with cocaine; 4) WT mice pretreated with L-NMMA 30 min prior to cocaine administration (iNOS-Inh). L-NMMA (NG-Methyl-L-arginine acetate salt, Millpore) is a non- selective iNOS inhibitor (Chung et al., 2021), that has been used in clinical trials to reduce ischemic lesion volume (Chung et al., 2021; Peterson et al., 1992) and to protect against neuronal injury. In preclinical studies, L-NMMA has also been shown to attenuate neurological dysfunction (Bagetta et al., 2004; Parmentier et al., 1999).

By integrating μOCT and MIP in this comprehensive study design, we aimed to gain insights into the role of iNOS in mediating the vascular effects of chronic cocaine exposure and to explore the potential therapeutic effects of iNOS inhibition in preventing or mitigating these outcomes.

2. Methods

2.1. Animals and experimental design

Inducible nitric oxide synthase knock out (iNOS-KO) mice and C57BL/6 wild type (WT) mice (Jackson Laboratory) at 8–10 weeks of age were used to assess the role of iNOS in cocaine-induced vascular changes. Mice were divided into four groups as shown in Fig. 1. Studies were approved by the Institutional Animal Care and Use Committee of Stony Brook University (Protocol number 252462 entitled “Cocaine-induced brain functional change using fluorescent and optical imaging”). All experimental procedures were conducted in accordance with the National Institutes of Health (NIH) Guidelines for Care and Use of Laboratory Animals.

Fig. 1. Experimental protocol and treatment timeline for saline and cocaine administration.

a) Timeline of chronic treatment and imaging sessions. All animals underwent cranial window implantation on Day −1. Mice received daily intraperitoneal (i.p.) injections of either saline or cocaine for 14 days. OCT imaging was performed on Day 0 (baseline) and Day 16 (24 h after final treatment), with three MIP imaging conducted at (Day 1, ~Day7, and Day 14) in the prefrontal cortex of mice to access cerebral vascular changes. b) Group1(Saline-WT): C57BL/6 mice, n = 6 received daily saline (0.7 ml/100 g/day, i.p.) and 0.1 ml i.v. saline during imaging. c) Group2 (Cocaine-WT): C57BL/6 mice, n = 5 received daily cocaine treatment (30 mg/kg/day, i.p.) and 1 mg/kg i.v. cocaine during imaging. d) Group3 (Cocaine-iNOS-KO): iNOS-Knock out mice, n = 6 received the same chronic and acute cocaine regimen as Group 2. e) Group4 (Cocaine-iNOS-Inh): C57BL/6 mice, n = 3 pretreated with L-NMMA (a non-specific iNOS inhibitor, 40 mg/kg/day, i.p.) 30 min prior to daily cocaine administration.

2.2. Implantation for optical imaging and chronic drug treatment

Before optical experiments, a cranial window (~2 ×2 mm²) was created over the PFC (A /P: +2.5; M/L: 0.5; D/V: −0.5 mm) for each animal to allow for optical imaging (Park et al., 2013). The PFC was chosen as this brain region has a crucial role in executive control and behavioral regulation (Menon and D’Esposito, 2022), and its disruption in addiction has been associated with loss of control and compulsive drug taking (Menon and D’Esposito, 2022; Jentsch and Taylor, 1999). Mice were anesthetized with inhalational 2 % isoflurane in oxygen and placed on a custom stereotaxic frame to minimize motion artifacts. The skull was carefully removed, and the exposed cortical surface was immediately covered with a sterile coverslip (4 ×3 mm²) and sealed with biocompatible glue. The respiration and body temperature were continuously monitored during surgery and imaging. After window implantation, the drug treatments for each group started as well as their image experiments as illustrated in Fig. 1. Specifically, Group1 (Saline-WT) were chronically treated with saline (0.7 ml/100 g/day, i.e. for 14 days), Group2 (Cocaine-WT) and Group3 (Cocaine-iNOS-KO) were chronically treated with cocaine (30 mg/kg/day, i.p. for 14 days) except on imaging days when they received an i.v. injection of cocaine; and Group4 (Cocaine-iNOS-Inh) were pretreated with L-NMMA (40 mg/kg/day, i.p.) 30 min prior to cocaine administration for 14 days. Dosages of cocaine use are chosen based on prior in-vivo optical imaging studies (Du et al., 2021; Chen et al., 2016b) and also these doses result in cocaine plasma levels consistent to those observed in cocaine abusers (Morrell et al., 2011).

2.3. System setup

2.3.1. μOCA imaging of vascular network before and after chronic drug exposure

To assess vascular changes after chronic cocaine exposure in iNOS-KO and WT mice with or without L-NMMA pretreatment, we performed in-vivo 3D imaging with μOCT (You et al., 2015) at baseline (Day 0) and post-treatment (Day16, i.e., 1 day withdraw after the final cocaine administration). For comparison, a vehicle group (daily saline treated) was imaged as well (Fig. 1). As illustrated in Supplemental Figure S1, the μOCT system employed an ultra-broadband light source (λ = 1310 nm; Δλ_FWHM≈200 nm) was used for illumination to achieve 2.5 μm axial resolution in tissue. The light beam exiting the sample arm was collimated to 5 mm, then steered transversely by a servo scanner (VM500, General Scanning), and focused through the cranial window by a near-infrared (NIR) achromatic doublet (f=18 mm/NA=0.25). The lateral resolution is determined by the effective numerical aperture (NA) of the achromat to be 3.2 μm. The backscattered light from mouse cortex and reference arm was recombined in the detection fiber connected to a custom spectrometer and detected by a fast line scan InGaAs CMOS camera (2048-pixels, GL2048, Sensors Unlimited), which was interfaced with an image workstation via a camera link card.

2.3.2. Dynamic imaging during chronic treatment

Our custom-built MIP (Chen et al., 2016b; Allen et al., 2019; Yuan et al., 2011; Gu et al., 2018) was able to capture reflected cortical images at two wavelengths λ₁= 568 nm and λ₂= 630 nm (Supplemental Figure S2a). These dual-wavelength measurements enabled the quantification of oxygenated-Δ[HbO₂] and deoxygenated-hemoglobin Δ[HbR] changes, thus resulting in changes in total hemoglobin (Δ[HbT]) from the PFC of mice at multiple time points during chronic cocaine treatment (Day1, ~Day7 and Day14). As illustrated in Supplemental Figure S2, light sources were delivered to the cortex with a time-sharing mode, alternating between the two wavelengths with an exposure time of 10 ms per channel. The reflected images captured at λ₁ and λ₂ were detected by a sCMOS camera and to extract relative changes in Δ[HbO₂] and Δ[HbR] in the mouse cortex. To assess the acute hemodynamic response to drug challenge, mice received an i.v. injection of either saline (0.1 ml) or cocaine (1 mg/kg) via the tail vein during imaging. Each imaging session lasted 50 min, including a 10-minute baseline followed by 40 min post saline or cocaine administration.

2.4. Image processing and quantitative analyses

2.4.1. Vascular diameter quantification

μOCA was reconstructed based on the voxel decorrelation between temporally adjacent OCT B-scans to extract speckle dynamics, thereby enabling the identification of vasculature from the surrounding static tissue. To access cocaine-induced vasoconstriction in the vascular network before and after chronic cocaine exposure, and to compare the potentially different changes between WT mice and iNOS-KO mice, the vessel diameters were quantified using a custom Matlab program based on a multi-scale vascular segmentation algorithm. Firstly, we normalized the image and applied the median filter to smooth the speckle noise. Next, the Frangi vesselness filtering (Frangi et al.) and the intensity-based method were combined used to detect vessels across multiple scales. Specifically, the vessels score for medium to small vessels was calculated based on the eigenvalue analysis of the Hessian matrix:

$$H_{I(x,y)}=\left[\begin{array}{cc}{\partial }^{2}I/\partial x^2& {\partial }^{2}I/\partial x\partial y\\ {\partial }^{2}I/\partial y\partial x& {\partial }^{2}I/\partial y^2\end{array}\right]$$ (1)

where ∂²I/∂x² and ∂²I/∂y² are the second derivative of image intensity I in x and y directions respectively, while ∂²I/(∂x∂y) represents the partial derivative. The filter scale varied from 1.0 to 3.0 with a step size of 0.25 to capture vessels of different sizes. Following vesselness computation, we applied the Adaptive Thresholding method (window size = 400, coefficient = −0.1) to segment small and medium-sized vessels based on the Frangi scores. To identify large vessels, we empirically used the intensity threshold of 100 (about 85th percentile) to binarize the large vascular area (as illustrated in Supplemental Figure S1c). Subsequently, morphological operations such as closing and opening were applied to remove segmentation artifacts. The final mask is fused including small, medium and large masks. The vessel diameter is measured using the Euclidean Distance Transformation, where each pixel is labeled with the distance to its closest background pixel (Supplemental Figure S1d₂). The vessel skeleton is generated using a morphological thinning algorithm (Supplemental Figure S1d₁) (Lam et al., 1992).

2.4.2. Microvascular density calculation

Microvascular density in the mouse PFC was assessed by generating a heating map, which was derived from μOCA image. A sliding window approach was employed to compute a spatial map of vascular density across the imaged region. First, we employed the Frangi vesselness filter (Frangi et al.) and thinning-based skeletonization algorithm (Lam et al., 1992) to generate the vessel masks (Supplemental Figure S1c) and skeleton map, respectively. Morphological dilation and erosion operations were applied to the skeleton map to repair discontinuities caused by speckle noise (Supplemental Figure S1c–d3). The local vascular density, or “Fill Factor” (FF) (You et al., 2017), was calculated for each pixel (x, y) as,

$$D_{(x,y)}=\frac{\Vert S_{(x,y,w)}\Vert }{w^2}$$ (2)

Where S_(x,y,w) is the image patch of size w= 60 centered at pixel (x, y) in the skeleton map S. The FF was computed within each patch, and this calculation was repeated in a sliding window across the entire image to generate a microvascular density map (Supplemental Figure S1d₄). Following that, the density map was normalized and smoothed with a Gaussian filter (σ = 3), improving the numerical stability and removing the boundary artifacts. Set of regions of interests (ROIs) were selected to calculate the maximum and mean vascular density of each image. The changes in these parameters before and after saline or cocaine exposure were subsequently quantified.

2.4.3. Hemodynamics changes in response to cocaine

To detect hemodynamic response to cocaine, each animal was imaged periodically during the chronic drug treatment (saline or cocaine) as shown in Fig. 1. During imaging, Group1 (Saline-WT) received a saline injection, while Group 2 (Cocaine-WT) and Group 3 (iNOS-KO) received cocaine (1 mg/kg, i.v.). The imaging protocol and signal processing steps are detailed in Supplemental Figure S2. For each animal, nine regions of interest (ROIs) were selected within the PFC to quantity hemodynamic changes, including changes in total hemoglobin (i.e., Δ[HbT]) and oxygenated hemoglobin Δ[HbO₂]) were calculated based on the following equation:

$$\left[\begin{array}{c}\Delta \left[Hb{O}_2(t)\right]\\ \Delta [HbR(t)]\end{array}\right]={\left[\begin{array}{cc}{\epsilon }_{Hb{O}_2}^{{\lambda }_1}& {\epsilon }_{HbR}^{{\lambda }_1}\\ {\epsilon }_{Hb{O}_2}^{{\lambda }_2}& {\epsilon }_{HbR}^{{\lambda }_2}\end{array}\right]}^{-1}\cdot \left[\begin{array}{l}\frac{\text{In}\left(R_{{\lambda }_1}(0)/R_{{\lambda }_1}(t)\right)}{L_{{\lambda }_1}(t)}\hfill \\ \frac{\text{In}\left(R_{{\lambda }_2}(0)/R_{{\lambda }_2}(t)\right)}{L_{{\lambda }_2}(t)}\hfill \end{array}\right]$$ (3)

where ${\epsilon }_{\text{HbO}2}^{\lambda }$, and ${\epsilon }_{\text{HbR}}^{\lambda }$ are the molar extinction coefficients for Δ[HbO₂] and Δ[HbR], and R_λ1(t), R_λ2(t) are the measured diffuse reflectance at these wavelengths. R_λ1(0), R_λ2(0) are their baseline values before cocaine. L_λ1(t), L_λ2(t) are their pathlengths. The total hemoglobin concentration change can be obtained by

$$\Delta [\text{HbT}]=\Delta \left[{\text{HbO}}_2\right]+\Delta [\text{HbR}]$$ (4)

This metric corresponds to the cerebral blood volume (CBV) change and serves as an indicator of local vascular response to drug challenge.

2.5. iNOS and VEGF measurements

Following the final optical imaging session, mice were euthanized for ex-vivo analysis. The PFC was dissected from the mouse brain and either fixed in 4 % formaldehyde or homogenized at 15,000 Hz for 15 s. Total iNOS expression was quantified by an iNOS ELISA kit (Abcam) with standard capture and detection antibodies and TMB-based colorimetric detection at 600 nm. VEGF levels were assessed by immunofluorescence microscopy on fixed brain slices using anti-VEGF antibodies, with fluorescence intensity quantified across experimental groups.

2.6. Statistics and reproducibility

Results are presented as means ± standard error (SEM). Comparisons between the four groups used One-way ANOVA followed by Tukey’s HSD post hoc. All statistical tests were performed using SigmaStat software (Systat Software Inc.), with P < 0.05 considered significant. Sample sizes (n = 6 per group) were determined based on previous in-vivo imaging publications and confirmed by a post hoc power analysis as shown in Fig. 1. Mice were randomly assigned to treatment groups. Image acquisition was not blinded due to protocol requirements. However, all subsequent image analysis and quantification were conducted by investigators blinded to group identity to minimize observer bias.

3. Results

We used MIP to image vascular and tissue hemodynamic responses (e.g., Δ[HbT] and Δ[HbO₂]) in PFC following acute saline (0.1 ml, i.v., Group 1 (Saline-WT)) or cocaine challenge (1 mg/kg, i.v.,) for both Group 2 (Cocaine-WT) and Group 3 (iNOS-KO). These measurements were conducted periodically during chronic saline or cocaine administration (on Days 1, ~7, and 14). We compared: 1) mean time courses of Δ[HbT] and Δ[HbO₂] following acute saline or cocaine injection; 2) peak amplitude of responses Δ[HbT] and Δ[HbO₂], and 3) integrated (area under curve) responses of Δ[HbT] and Δ[HbO₂]. In parallel, μOCT was used to assess vascular morphological changes (e.g., vessel diameter and microvascular density) before (Day 0) and after (Day 16) exposure to chronic saline or cocaine treatment. Four experimental groups were included: Group 1 (Saline-WT), Group 2 (Cocaine-WT), Group 3 (Cocaine-iNOS-KO) and Group 4 (Cocaine-iNOS-Inh). To compare these 4 groups of animals, we quantified changes in: 1) mean vessel diameter; 2) mean vessel density and 3) maximum vessel density.

3.1. Vascular [HbT] and tissue oxygenation [HbO₂] are temporarily decreased by the first acute cocaine dose in both WT and iNOS-KO mice

Acute cocaine administration on Day1 reduced Δ[HbT] and Δ[HbO₂] in PFC of both WT and iNOS-KO mice. Fig. 2a–c show representative spatiotemporal images of Δ[HbT] concentration over time following saline (Group1) or cocaine (Group2 and Group3) injection, which demonstrate the spatiotemporal Δ[HbT] decreases induced by cocaine for both Group2 (Fig. 2b i–v) and Group3 animals (Fig. 2c i–v).

Fig. 2. Hemodynamic responses on Day1 across experimental groups.

a-c) Representative images showing dynamic changes (ii-v) in response to saline or cocaine and Δ[HbT] maps at t = 0, 2, 10, 40 min post-injection. d) Mean time courses of Δ[HbT] in response to saline or cocaine challenge, showing a slightly decrease induced by saline (blue line, n = 6) followed by return to baseline immediately, but long-persistent decreases induced by cocaine in both WT animals (green line, n = 5) and iNOS-KO animals (purple line, n = 6). e₁) Comparison of peak Δ[HbT] decreases (%), showing significant differences between both cocaine treated mice and saline treated controls (P*<0.001). e₂) Comparisons of Δ[HbT] decay rate (%/min) showing significant difference between Cocaine-WT and control (P*=0.010) as well as Cocaine-iNOS-KO animals over the controls (P*=0.002). f) Mean Δ[HbO₂] changes with time in response to saline or cocaine, showing cocaine-induced Δ[HbO₂] decrease in cortical tissue of cocaine animals around 36 min followed by a recovery to baseline. Cocaine-iNOS-KO animals have a long persistent decrease of around 20 % during the whole experimental time. g₁) Comparison of peak Δ[HbO₂] reduction showing significant difference between controls and cocaine (P*<0.001) as well as cocaine treated iNOS-KO animals (P*<0.001). g₂) Comparisons of Δ[HbO₂] decay rate also shows cocaine WT and iNOS-KO have a greater reduction compared to saline controls. All error bars are presented as means ± SEM.

Fig. 2d summaries the group averaged Δ[HbT] time courses. Both WT and iNOS-KO mice exhibited significant reduction in Δ[HbT] following cocaine injection while Saline-WT showed no changes after saline injection. Specifically, iNOS-KO mice had a maximum Δ[HbT] decrease of (−21.60 ± 1.906 %) (n = 6), remaining below baseline for the entire 40 min imaging period. WT mice showed a decrease of (–23.36 ± 2.55 %) (n = 5), followed by a gradual recovery starting 5–8 min post cocaine. Meanwhile, saline controls showed a non-significant decrease (−3.519 ± 1.576 %) (n = 6), returning quickly to baseline. Comparisons for peak Δ[HbT] decreases are summarized in Fig. 2e₁, One-way-ANOVA revealed significant differences between the control and both cocaine-treated groups (WT and iNOS-KO), with P*< 0.001 for each comparison. The mean Δ[HbT] decay rates (integrated reactivity, Δ[HbT]%/min) are shown in Fig. 2e₂, for controls, Δ[HbT]%/min was (−2.202 ± 0.5503 %), which differed compared to Cocaine-WT (−15.99 ± 3.180 %) (P*=0.010) and Cocaine-iNOS-KO (−19.65 ± 3.814 %) (P*=0.002) groups. These findings indicate that the first acute dose of cocaine significantly reduced cerebral blood volume (as reflected by Δ[HbT]) in both WT and iNOS-KO animals, with no statistically significant difference between the two groups.

Fig. 2f displays their changes in Δ[HbO₂]. Cocaine injection immediately reduced mean Δ[HbO₂] to (−21.71 ± 2.752 %) in WT, followed by a gradual recovery at t = 36 min. For iNOS-KO mice, Δ[HbO₂] also decreased to (−21.05 ± 3.101 %), with no recovery observed over the full 40-minute period. Controls Δ[HbO₂] was slightly decrease to (−2.956 ± 1.244 %) and returned to baseline immediately. Fig. 2g₁ summarizes peak Δ[HbO₂] reductions. Similar to Δ[HbT], both cocaine-treated groups differed significantly in Δ[HbO₂] from controls (P*<0.001). The mean Δ[HbO₂] decay rates are summarized in Fig. 2g₂, showing that compared to saline injection (−1.081 ± 1.112 %), cocaine injection in WT (−11.40 ± 3.412 %) and iNOS-KO mice (−16.17 ± 3.296 %) resulted in significantly greater reduction of mean Δ[HbO₂]. These results show that acute cocaine induces marked decreases in tissue oxygenation, which persists longer in iNOS-KO mice.

3.2. Cocaine-induced [HbT] and [HbO₂] reduction are attenuated in iNOS-KO mice following sub-chronic cocaine treatment (~7 days)

After approximately 7 days of daily i.p. cocaine treatment, the acute hemodynamic responses to i.v. cocaine (1 mg/kg) were reassessed in all animals. Supplemental Figure S3a–c shows representative baseline raw images and time-lapse images of Δ[HbT] changes following saline in Group1 or cocaine in Group2 and Group3. These images showed that cocaine induced spatiotemporal decreased in Δ[HbT] in PFC, with greater reductions observed in WT compared to iNOS-KO mice.

Figure S3d showed mean Δ[HbT] change. Acute cocaine led to an immediate reduction in Δ[HbT], with peak changes of (−30.25 ± 3.100 %) in WT and (−20.28 ± 2.039 %) in iNOS-KO mice. In contrast, the control group exhibited a minor reduction after intravenous saline injection (−6.142 ± 1.760 %). Comparison of the peak amplitudes for these three groups are summarized in Figure S3e₁, One-way ANOVA indicated that both cocaine treated groups differed significantly from controls (P*<0.001 for WT; P*=0.001 for iNOS-KO), and that the reduction in WT mice was significantly greater than in iNOS-KO mice (P*=0.009). Analysis of the Δ[HbT] decay rate (Δ[HbT]%/min) further supported these findings shown in Figure S3e₂, with values of (−25.20 ± 2.517 %) in WT, (−16.50 ± 1.946 %) in iNOS-KO and (−3.822 ± 1.380 %) in controls (P*<0.001 for both cocaine-treated groups vs. controls; P*=0.008 for WT vs. KO).

Tissue oxygenation responses showed a similar pattern. As shown in Figure S3f, Δ[HbO2] decreased immediately after cocaine administration in both treated groups, reaching (−22.19 ± 2.984 %) in WT and (−16.50 ± 1.271 %) in iNOS-KO. While WT mice displayed partial recovery over the imaging period, both cocaine treated mice remained below baseline throughout. Control animals showed a slight decrease to (−4.081 ± 0.664 %) after saline injection. Summary of peak reductions are shown in Figure S3g₁, which showed that controls differed from WT (P*<0.001) and iNOS-KO (P*=0.001), respectively. Meanwhile, there was no statistical difference between WT and iNOS-KO mice. However, analysis of Δ[HbO₂] decay rates (Figure S3g₂) revealed a significant attenuation in iNOS-KO mice (−12.74 ± 1.438 %) compared to WT (−16.76 ± 1.377 %, P*=0.034), suggesting that recovery from cocaine-induced oxygenation loss was slower in the absence of iNOS.

3.3. Chronic cocaine exposure (2 weeks) induced persistent reductions in vascular [HbT] and tissue oxygenation [HbO₂] in WT mice which lessened in iNOS-KO mice

To evaluate the long-term effects of cocaine on cerebral hemodynamics, we assessed changes in Δ[HbT] and Δ[HbO₂] following acute cocaine injection (1 mg/kg, i.v.) after 14 days of chronic cocaine treatment. Fig. 3a–c showed representative baseline images and images of the percent changes in Δ[HbT] over time at different time points (t = 2, 10 and 40 min) following saline (For Saline-WT, Group1) or cocaine (Cocaine-WT(Group2) and Cocaine-iNOS-KO (Group 3)), respectively.

Fig. 3. Hemodynamic responses on Day14 between Saline_WT, Cocaine_WT and Cocaine_iNOS-KO mice.

a-c) Representative images showing dynamic changes (ii-v) in response to saline (0.1 ml, i.v.) in Group 1 (Control animals) or cocaine (1 mg/kg, i.v) in Group 2 (Cocaine animals) and Group 3 (Cocaine-iNOS-KO animals) at t = 0, 2, 10, 40 min. d) Mean time courses of Δ[HbT] in response to the saline or cocaine challenge, showing a slightly decrease reduced by saline (blue line, n = 6), but long-persistent decreases induced by cocaine in both cocaine animals (green line, n = 5) and Cocaine-iNOS-KO animals (purple line, n = 6). e₁) Comparison of peak Δ[HbT] decreases between Group 1, Group 2 and Group 3, showing significance difference between cocaine animals (P*<0.001) as well as Cocaine-iNOS-KO (P*=0.013) and control group. e₂) Comparisons of Δ[HbT]% decrease per minute between Group 1, Group 2 and Group 3, showing significant difference between cocaine animals and control (P*=0.019) as well as Cocaine-iNOS-KO animals over the controls (P*<0.001). Meanwhile, comparisons between cocaine WT mice and Cocaine-iNOS-KO animals also show significant difference. (P*=0.014) f) Mean Δ[HbO₂] changes with time in response to saline (0.1 ml, i.v) from Group 1 (blue dash line, n = 6), and cocaine from Group 2 (green dash line, n = 5) and Group 3 (purple dash line, n = 6) animals, showing cocaine-induced Δ[HbO₂] decrease in cortical tissue of Cocaine-WT and Cocaine-iNOS-KO group animals decrease around 25 % during after cocaine administration. g₁) Comparison of peak Δ[HbO₂] decreases between Group 2 and Group 3 showing no significant difference. g₂) Comparisons of Δ[HbO₂] % decrease per minute showing significant difference between cocaine animals (P*=0.003) as well as iNOS_KO (P*=0.006) over controls. All error bars are presented as means ± SEM.

Fig. 3d showed the mean Δ[HbT] changes, which reveals a rapid and sustained decrease in total blood volumes in both cocaine treated groups. At 2 min post-injection (Δt = 2 min), Δ[HbT] declined to (−26.22 ± 3.715 %) in WT mice and to (−18.54 ± 1.866 %) in iNOS-KO mice, both remaining below baseline throughout the 40-minute recording period. Control animals had a moderate decreased to (−4.397 ± 1.294 %) after saline injection. Comparison of peak Δ[HbT] values across groups (Fig. 3e₁), showed no significant difference between WT and iNOS-KO mice. However, integrated reactivity of Δ[HbT] (%/min) comparison between WT mice (−23.29 ± 1.933 %) and iNOS-KO mice (−13.93 ± 1.140 %) was significant (P*=0.014), suggesting attenuated vascular reactivity in the absence of iNOS.

Changes in Δ[HbO₂] followed a similar pattern. Fig. 3f showed Δ[HbO₂] decreased immediately after cocaine injection to (−19.10 ± 2.732 %) in WT and (−15.74 ± 2.972 %) in iNOS-KO mice, with both remaining below baseline throughout the imaging period (t = 40 min). Saline injected controls showed a small decrease (−2.056 ± 0.761 %) that returned to baseline by 28 min. Peak Δ[HbO₂] comparisons shown in Fig. 3g₁, indicated no differences between WT and iNOS-KO mice. However, Δ[HbO₂] decay rates analysis (Fig. 3g₂) indicated that the control group (−2.893 ± 0.4838 %) differed significantly from Cocaine-WT (−15.79 ± 2.829 %, P*=0.003) and iNOS-KO group (−13.88 ± 2.147 %, P*=0.006). while the difference between the two cocaine-treated groups was not statistically significant. Together, these results demonstrate that after 14 days of chronic cocaine exposure, Δ[HbT] remained significantly attenuated in iNOS-KO mice compared to WT, whereas Δ[HbO₂] reduction persisted in both groups.

3.4. Sensitization of hemodynamic responses after 2 weeks of cocaine were reduced in iNOS-KO compared to WT mice

To evaluate the sensitization of hemodynamic changes with chronic exposure, we compared the magnitude of the responses to cocaine on Day 14 relative to Day 1. Fig. 4 shows the decay rate of Δ[HbT] (%/min) and Δ[HbO₂] (%/min) after acute cocaine on Day 1 (yellow bars) and Day 14 (orange bars) for Saline-WT (Group 1), Cocaine-WT (Group 2), and Cocaine-iNOS-KO (Group 3) animals. The corresponding response ratios defined as the percent change from Day 1 to Day 14 ((Day14-Day1)/Day1 *100 %), displayed on the 2nd Y-axis (right side) in Figs. 4a, 4b for Δ[HbT] and Δ[HbO₂], respectively. In Cocaine-WT mice, the Δ[HbT] decay rate increased from (−15.99 ± 3.180 %/min) on Day 1 to (−23.29 ± 2.932 %/min) on Day 14, representing a (Δ=62.49 ± 9.460 %) increase in reactivity. In contrast, Cocaine-iNOS-KO mice showed a reduced response, with decay rates decreasing from (−19.65 ± 3.814 %/min) to (−13.93 ± 1.140 %/min), corresponding to a (Δ= −29.08 ± 5.803 %) change. These differences between WT and iNOS-KO groups were statistically significant (P*< 0.001). Similarly, in Cocaine-WT animals, Δ[HbO₂] decay rates increased from (−11.40 ± 3.412 %/min) (Day 1) to (−15.80 ± 2.830 %/min) (Day 14), yielding a (Δ=54.67 ± 18.37 %) change. Conversely, in Cocaine-iNOS-KO mice, decay rates decreased from (−16.17 ± 3.296 %/min) to (−13.88 ± 2.147 %/min), reflecting a (Δ= 25.01 ± 9.884 %) change. The difference between these two groups was also statistically significant (P* =0.022). In contrast, saline controls exhibited no significant difference in Δ[HbT] or Δ[HbO₂] responses between Day 1 and Day 14, confirming the stability of baseline vascular reactivity in the absence of cocaine. Together, these results demonstrate that chronic cocaine exposure worsens vascular and oxygenation response in WT mice, whereas iNOS deletion results in an attenuated adaptation, particularly for Δ[HbT].

Fig. 4. Comparison of cocaine-induced Δ[HbT] and Δ[HbO₂] responses between Day1 and Day14 for Saline-WT, Cocaine-WT and Cocaine-iNOS-KO mice.

a) Bar plots comparing the rate of change in Δ[HbT] (%/min, left y-axis) and relative change (% in Day 14 over Day 1, right y-axis) in Saline-WT (Group1), Cocaine-WT (Group 2), and Cocaine-iNOS-KO (Group 3), respectively. Cocaine-WT mice exhibited a significantly greater increase in Δ[HbT] reactivity from Day 1 to Day 14. In contrast, iNOS-KO mice showed an attenuated response over time, with a negative ratio between Day 14 and Day 1, which has significantly difference compared to Cocaine-WT (P*<0.001). b) Bar plots showing Δ[HbO₂] (%/min, left y-axis) and relative change (%) across the experimental groups. While Cocaine-WT mice demonstrated increased oxygenation reactivity on Day 14 compared to Day1, iNOS-KO mice showed reduced responses. The difference between Cocaine-WT and iNOS-KO mice in Δ[HbO₂] changes was statistically significant (P*=0.022).

3.5. Cerebral vasoconstriction following chronic cocaine was reduced in iNOS-KO mice and iNOS-Inh mice

To evaluate iNOS’s involvement in cocaine-induced vasoconstriction, we measured the vascular diameters across four experimental groups using μOCA before (Day0) and after (Day16) chronic treatment. Fig. 5a–d presents representative vascular images and their corresponding diameter map from Saline-WT (a₁,a₂), Cocaine-WT (b₁,b₂), Cocaine-iNOS-KO (c₁,c₂) and Cocaine with L-NMMA pretreatment (Cocaine-iNOS-Inh) mice. Each row shows the raw vascular image (i), the extracted vascular map (ii), and the diameter map (iii). As expected, in saline controls, there was no obvious vasoconstriction between Day 0 and Day 16 (a₃: 56.22 ± 4.196 μm vs 53.06 ± 3.981 μm, relative diameter changes Δ= −3.851 ± 1.117 %, m=20), indicating vascular stability. In contrast, Cocaine-WT mice exhibited robust vasoconstriction, with diameters decreasing (from 60.47 ± 4.255 μm to 46.43 ± 3.160 μm), corresponding to a significant relative reduction (Δ =−22.58 ± 1.54 %), as shown in Fig. 5b₃. Importantly, the magnitude of vasoconstriction was significantly attenuated in iNOS-KO and iNOS-Inh mice. In iNOS-KO mice, vascular diameters decreased from (c_3:58.86 ± 3.214 μm to 49.71 ± 2.510 μm, Δ=−15.15 ± 1.409 %). Similarly, iNOS-Inh mice showed a reduction (d_3: 47.74 ± 2.451 μm to 39.74 ± 2.165 μm, Δ=−14.90 ± 1.965 %). Group comparisons of vasoconstriction responses are summarized in Fig. 5e. Statistical analysis revealed that cocaine-induced vascular diameter reduction in WT mice was significantly greater than in both iNOS-KO (P*=0.002) and iNOS-Inh (P*=0.002) groups. These findings strongly support a role for iNOS in mediating cocaine-induced vasoconstriction and indicated that inhibition of iNOS mitigates vascular constriction following chronic cocaine exposure.

Fig. 5. Vascular diameter changes comparison between Saline-WT, Cocaine-WT, iNOS-KO and iNOS-Inh mice.

a-d) Representative μOCA images and corresponding vessel segmentation and quantitative diameter maps from Day0 (baseline) and Day16 for four experimental groups: a) Saline-WT; b) Cocaine-WT; c) Cocaine-iNOS-KO; d) Cocaine-iNOS-Inh mice. Each group includes: i) raw μOCA images; ii) binary vascular maps; iii) quantitative diameter maps, scale bar indicates diameter in mm, red arrows indicate vasoconstriction, and blue arrows indicate mild vasodilation. a₃-d₃) Quantitative comparisons of average vessel diameter (μm) before (grey) and after chronic treatment. In the absence of cocaine (Saline-WT), no significant diameter change observed (a₃, P = 0.588). In contrast, chronic cocaine exposure led to significant vasoconstriction in Cocaine-WT (b₃, P*=0.011), iNOS-KO (c₃, P*=0.031) and iNOS-Inh (d₃, P*=0.016) groups. e) Comparison of relative vascular diameter changes in all four groups showing that Cocaine-WT mice exhibited a greater reduction in vessel diameter (−22.58 ± 1.542 %). This effect was significantly attenuated in iNOS-KO (P*=0.002) and iNOS-Inh (P*=0.002). Both treatment groups differed significantly from Saline-WT (P*<0.001). Red and blue dots represent arteries and veins, repressively. Color coded in blue, green, purple and orange represent saline-WT, cocaine-WT, cocaine-iNOS-KO, and cocaine-iNOS-Inh, respectively.

3.6. Chronic cocaine caused enhanced angiogenesis in iNOS-KO and iNOS-Inh mice

To assess angiogenic responses to chronic cocaine exposure, microvascular density changes were quantified from density heatmaps derived from μOCA images. For each animal, five randomly selected regions of interest (ROIs) were analyzed before (Day 0) and after (Day 16) treatment. Fig. 6a–d shows microvascular density maps extracted from Saline-WT (a₁,a₂), Cocaine-WT(b₁,b₂), Cocaine-iNOS-KO (c₁,c₂) and Cocaine-iNOS-Inh (d₁,d₂) mice. Corresponding density heatmaps (i) and vascular network maps (ii) illustrate spatial microvascular distribution across the PFC. Quantitative analysis of mean microvascular density showed no significant change in saline control group, with values increasing only modestly from (a_3: 0.0634 ± 0.033 to −0.0662 ± 0.003, relative mean density changes Δ=5.048 ± 2.351 %). In contrast, chronic cocaine significantly increased mean microvascular density in Cocaine-WT mice from (b₃:0.0518 ± 0.0029 to−0.0630 ± 0.0041, Δ=21.39 ± 3.825 %, P*=0.001 vs. controls). Maximum local density also increased significantly (P*=0.02), suggesting that vasoconstriction induced ischemia may have triggered compensatory angiogenesis. Notably, the increase in microvascular density was further enhanced in iNOS-KO and iNOS-Inh mice. In iNOS-KO mice, mean density rose from (c₃: 0.043 ± 0.0024 to −0.0719 ± 0.0036) corresponding to (Δ=69.83 ± 12.07 %) increase (P*<0.001 vs. Cocaine-WT). Similarly, iNOS-Inh mice exhibited an increase from (d₃: 0.041 ± 0.0025 to −0.063 ± 0.004, Δ=50.39 ± 4.450 %, P*=0.030 vs. Cocaine-WT). However, the maximum microvascular density in iNOS-KO and iNOS-Inh mice did not significantly differ from that of WT mice. Meanwhile, we quantified VEGF expression data as shown in Supplemental Figure S5. VEGF levels were significantly elevated in iNOS-KO (84.04 ± 0.9252 %) mice compared to saline controls (39.26 ± 16.04 %) (P* = 0.026) and showed a trend toward further increase compared to cocaine-WT mice (54.01 ± 2.089 %) (P* = 0.05). Taking together, these results indicate significant angiogenesis with chronic cocaine that were enhanced in iNOS-KO and L-NMMA pretreated mice, which could reflect compensatory responses to counteract inadequate perfusion from vasoconstriction.

Fig. 6. Microvascular Density changes comparison between Saline-WT, Cocaine-WT, iNOS-KO and iNOS-Inh mice.

a-d) Representative vascular density heatmap, binary vascular masks, and skeletonized vessel maps before (Day0) and after (Day16) in four groups: a) Saline-WT; b) Cocaine-WT; c) Cocaine-iNOS-KO; d) Cocaine-iNOS-Inh mice. For each group, subpanels show: i) Vessel density heatmap computed using a sliding-window algorithm from μOCA images; ii) binary vascular segmentation; iii) vascular skeleton image. a₃-d₃) Comparisons of mean vascular density (a.u.) before and after treatment. In the absence of cocaine (Saline-WT), there was no significant changes observed in vessel density over time (a_3, P = 0.580). In contrast, chronic cocaine exposure led to a moderate but significant increase in Cocaine-WT (b₃, P*=0.044). This angiogenic response was markedly enhanced in mice lacking iNOS (Cocaine-iNOS-KO, c₃, P* < 0.001) or receiving pharmacological iNOS inhibition (Cocaine-iNOS-Inh, d₃, P* < 0.001). e) Summary of relative changes in mean microvascular density (%) across groups. Both iNOS-KO and iNOS-Inh mice showed greater increases than Cocaine-WT (P*<0.001, P*=0.030, respectively). f) Changes in maximum local vessel density (%), all cocaine-treated groups increased significantly compared to Saline-WT. However, cocaine induced max density increase in iNOS-KO and iNOS-Inh mice were not significantly different from the Cocaine-WT.

4. Discussion

In this study, we demonstrated that chronic cocaine caused significant vasoconstriction in the PFC of WT mice, and that this effect was reduced in iNOS-KO and in iNOS-Inh mice. We further showed that chronic cocaine increased microvascular density, reflecting angiogenesis. Notably, this angiogenic effects were enhanced in both iNOS-KO and iNOS-lnh mice, which could reflect compensation to counteract inadequate perfusion from cocaine induced vasoconstriction. The enhanced angiogenesis in iNOS-KO and iNOS-Inh mice implicates iNOS in mediating cerebrovascular response to cocaine and identify it as potential medication target to remediate adverse consequences of cocaine on CBF and ischemia. Our result corroborates prior findings from our and other groups that chronic cocaine exacerbates the vasoconstrictive and hypoperfusion effects cocaine. Meanwhile, our longitudinal study design allowed us to track the dynamic changes triggered by continuous cocaine administration at Day1, ~Day7, Day14. We observed acute cocaine induced significant blood volume Δ[HbT] and tissue oxygenation Δ[HbO₂] reductions. Although iNOS-KO mice also exhibited reductions in Δ[HbT] and Δ[HbO₂] after cocaine, the integrated Δ[HbT] changes on Day 14 were significantly attenuated compared to Day 1 and lower than in WT mice (P*<0.001). Together with the observation of reduced vasoconstriction in iNOS-KO mice these results indicated that iNOS is involved in regulating global vascular tone and CBF responses. Moreover, they indicate that iNOS contributes not only to the acute hemodynamic effects of cocaine but also to the adaptive cerebrovascular changes that occur with chronic exposure.

4.1. iNOS regulation of vascular tone

Nitric oxide (NO) is a molecule involved in the regulation of blood flow, vasodilation, neuronal and glial activity (Wierońska et al., 2021) and is a crucial contributor to the pathological changes after stroke (Miki et al., 1977). The role of NOS (including nNOS, eNOS, and iNOS) as an inflammatory response has also been extensively studied (Tripathi et al., 2007). iNOS can be synthesized by multiple cell types and is usually expressed only when cells are stimulated or activated. iNOS expression in the brain can be induced by neurotoxin, inflammation, ischemia, and trauma; and iNOS expression by brain glial cells has been linked to various neurological and neurodegenerative diseases (Nakamura et al., 2012; Ghasemi and Fatemi, 2014; Galea et al., 1992). In a parallel study we measured iNOS expression in the mouse brain using sandwich ELISA and western blot and immunofluorescence microscopy to measure the number of activated astrocytes. The results which will be reported in a separate paper indicated that 2-week cocaine treatment induced a significant decrease in astrocyte numbers, as well as total iNOS expression. We also conducted in-vitro experiments in cultured human cortex astrocytes to investigate the signaling pathway involved and showed that cocaine significantly increased astrocyte iNOS expression though it did not affect NO production. We did not measure the expression of other NOS isoforms (eNOS and nNOS), which is a limitation. Nevertheless, previous studies reported that eNOS expression can increase in the absence of iNOS after ischemic injury (Forstermann and Sessa, 2012), and nNOS upregulation was observed when iNOS activity was reduced highlighting the plasticity and cross-regulation within the NOS family (Pautz et al., 2010).

4.2. L-NMMA inhibition of iNOS

Although L-NMMA is a non-selective NOS inhibitor that suppresses all three isoforms (iNOS, eNOS, and nNOS) (Kithas et al., 2020; Knowles and Moncada, 1994), several studies have demonstrated that under inflammatory or activated conditions, it preferentially inhibits iNOS. For example, L-NMMA significantly suppressed iNOS-mediated NO production in cytokine-stimulated endothelial and macrophage cells (Brosnan et al., 1997; Merenzon et al., 2023). In preclinical and clinical studies, L-NMMA has been used as an effective pharmacological tool to target iNOS in various disease models, including cancer and cardiovascular dysfunction (Merenzon et al., 2023; Granados-Principal et al., 2015). To validate iNOS suppression, we included iNOS expression data from the mouse PFC (Supplemental Figure S4) that shows undetectable iNOS expression in iNOS-KO mice and significantly reduced in L-NMMA treated mice compared to controls. These results verify the efficacy of both genetic and pharmacological inhibition strategies. While several more selective iNOS inhibitors such as 1400 W or L-NIL have been developed, their in-vivo use remains limited due to poor brain permeability, shorter half-life, or lack of approved systemic delivery routes in small-animal models (Underly and Shih, 2020; Minhas et al., 2020). Therefore, in this study, we applied a dual approach pharmacological inhibition using L-NMMA and genetic knockout of iNOS, which provides complementary evidence for iNOS’ involvement in cocaine-induced vascular responses.

While our results suggest that iNOS inhibition could be therapeutically valuable in mitigating cocaine induced neurovascular dysfunction, the possible adverse effects associated with long-term inhibition of iNOS need to be considered. iNOS plays a crucial role in host defense, particularly in innate immune responses against bacterial and parasitic infections (Zamora et al., 2000; Shen et al., 2022). Prolonged iNOS inhibition may impair microbial clearance, increase susceptibility to infections, and interfere with immune regulation. Additionally, iNOS deficiency has been shown to delay wound healing and promote excessive scar formation in skin injury models (Zamora et al., 2000; Farahani et al., 2025), and to exacerbate fibrosis in chronic liver disease models (Nozaki et al., 2015). Therefore, although iNOS inhibition may be beneficial for short term use, its prolonged use must be cautiously considered.

4.3. iNOS influence calcium channel activity

Cocaine has been widely reported to alter calcium homeostasis in PFC, leading to increased intracellular Ca²⁺ levels and contributing to excitotoxicity and neurovascular dysfunction (Du et al., 2006). These alterations might influence iNOS activity via modulation of NMDA receptor activity (Iadecola et al., 1997; Lipton et al., 1998) and voltage-gated calcium channels (Tozer et al., 2012). Previous studies demonstrated that astrocytic iNOS expression interfered with NMDA receptor activity, which led to greater Ca²⁺ influx and cell death, whereas iNOS inhibition significantly reduced neuronal damage (Mendoza-Baumgart et al., 2004). Moreover, iNOS activation has been shown to modulate L-type calcium channel activity through S-nitrosylation and the NO/cGMP signaling pathway, leading to enhanced glutamate release and increased Ca²⁺ accumulation in PFC neurons (Swanson et al., 2001; Awumey et al., 2013). Furthermore, when under cocaine treatment, upregulated iNOS leads to excess NO production and oxidative stress, which might further increase L-type channel activity and raise Ca²⁺ load (Agharahimi et al., 2021). In summary, the inhibition of iNOS might limit calcium accumulation and attenuate cocaine induced effects in the PFC.

4.4. Cocaine-induced vasoconstriction

Our findings showed cocaine decreased blood volume (HbT) and triggered vasoconstriction in vessels of all sizes. While, most studies of vasoconstriction report on diameter reductions in arteries some have reported vasoconstriction in veins (Shen et al., 2013). We observed that cocaine induced vascular diameter decreases in arteries veins and capillaries, which could underlie our prior findings of cerebral ischemia and TIA in rodents with chronic cocaine (Ren et al., 2012; You et al., 2014) and account for the clinical findings of increased risk for TIA and cerebral strokes in cocaine misusers (You et al., 2017; Du et al., 2020b).

4.5. Cocaine-induced divergence between HbT and tissue oxygenation

The decreases in blood volume and tissue oxygenation in the PFC of Cocaine-WT animals treated chronically with cocaine is in agreement with clinical observations of decreased CBF in the cortex, particularly in the PFC, of individuals who misuse cocaine (Volkow et al., 1988). In our study, we observed a significant decrease in cerebral blood volume following acute cocaine administration in both Cocaine-WT and Cocaine-iNOS-KO mice, and the decreases in Δ[HbT] persisted for at least 40 min. However, Δ[HbO₂], which was also decreased, recovered within 36 min followed by an overshoot above baseline in Cocaine-WT whereas for Cocaine-iNOS-KO the duration of the decreased Δ[HbO₂] was similar to their Δ[HbT] changes. The temporal mismatch between Δ[HbO₂] and Δ[HbT] observed in WT mice following cocaine exposure was not evident in iNOS-KO mice. This divergence might be protective to prevent ischemia in the WT and implicates iNOS involvement in the overshoot process, which was not observed in iNOS-KO in whom the prolonged duration of ischemia might promote enhanced angiogenesis as observed in iNOS-KO and iNOS-Inh mice.

The quantitative capability of our imaging tools with their high temporal and spatial resolutions allowed us to monitor dynamic changes in cerebrovascular networks. Our results clarify that cocaine induced reductions in CBF are due to cocaine-induced vasoconstriction. Our longitudinal (Day1, ~Day7, Day14) imaging revealed that cocaine-induced blood volume reductions intensified with chronic cocaine exposure but not observed from [HbO₂]. Specifically, Δ[HbT] decrease was sensitized with repeated administration in Cocaine-WT mice, whereas this was not the case in iNOS-KO mice. iNOS-KO mice showed prevented sensitization effect might due to NO synthesis has an effect in the vascular tone regulation of cerebral vasculature (Faraci, 1993; Kitazono et al., 1996) with mild dilating effects on the pial circulation under physiological conditions (Haberl et al., 1991; McKenna et al., 1994). Previous studies showed an inhibitory effect of vasoconstricting responses in the rat middle cerebral artery through a mechanism that involves iNOS activation (Alonso et al., 1998). Though mechanisms are unclear, there is evidence that norepinephrine (NE) and L-type Ca²⁺ channels may play a role in the sensitization of cocaine-induced vasoconstriction (Degoulet et al., 2016; Chen et al., 2013; Laporte and DeRoth, 1997; Kalsner, 1993).

4.6. Cocaine-induced increase in microvascular density

Longitudinal imaging allowed us to capture the increases in microvascular density following chronic cocaine, which might reflect angiogenesis. Indeed there is evidence that ischemia stimulates angiogenesis (Beck and Plate, 2009), which suggests that the microvascular density increase we observed is likely a consequence of cocaine-induced ischemia.

Revascularization after ischemic stroke plays an essential role in post-cocaine recovery (Fan et al., 2010). A previous longitudinal two-photon study on a mouse stroke model discovered that the newly formed capillary segments tended to grow toward the ischemic zone and the regenerated microvasculature became more aligned with the direction of ischemic center (Schrandt et al., 2015). For this work, we established a new strategy for microvasculature density analysis that allowed us to visualize and quantify local vascular density changes induced by cocaine without the need of an exogenous contrast agent as required for two photon microscopies.

We recently reported blood flow redistribution from superficial to deep cortical layers of mice exposed to chronic cocaine. The increases in vessel density including that in iNOS-KO mice and L-NMMA pretreated mice might reflect physiologically compensatory responses to prevent further hypoxia due to vasoconstriction. The enhanced angiogenesis with chronic cocaine exposure in iNOS-KO and L-NMMA treated mice compared to WT mice, indicates that blocking iNOS might promote increases in vessels density. Though the mechanisms of chronic cocaine triggered angiogenesis are unclear, VEGF has been implicated (Krupinski et al., 1994b; Wei et al., 2001), including in cocaine misusers (Strong et al., 2003). We quantified the VEGF expression levels (Figure S5), which were significantly elevated in the iNOS-KO group compared to saline controls (P*=0.026) and showed a trend toward further increase compared to cocaine-WT mice (P*= 0.05). Since, VEGF is a potent angiogenic factor involved in blood vessel growth in ischemic diseases (Marti et al., 2000), increases in VEGF could underlie the angiogenesis we observed in animals chronically exposed to cocaine. Though VEGF triggers endothelial cell proliferation and enhances vascular permeability in nascent vessels, newly formed vessels are immature and leaky (Zhang et al., 2002), which could explain why CBF remained decreased in the cocaine exposed animals despite the increases in vessel numbers. Previous studies also reported that hypoxia can trigger VEGF pathway activation in astrocytes (Weidemann et al., 2010). Thus, it is possible that vascular endothelial cells were not the only cells that contributed to the observed increase in VEGF expression following chronic cocaine treatment, and likely astrocytes could have contributed as well. However, the number of micro vessels filled with blood cells was significantly lower in the infarcted hemispheres, and the functionality of the new cerebral vessels following an ischemic event is unclear (Hayashi et al., 2003). Nevertheless, more attention is needed to investigate the role of angiogenesis, which could determine neuronal survival after cerebral ischemia, in the recovery of cocaine misusers.

The enhanced adaptations in blood vessels that we observed in the iNOS-KO mice and in mice pretreated with L-NMMA, is clinically relevant for it suggest that inhibition of iNOS might help accelerate brain recovery following cocaine-induced ischemia (Kusaka et al., 2005; Marti, 2005). The sensitized reactivity of cerebral blood vessels to chronic cocaine is also clinically relevant for it indicates that chronic cocaine users are at increased risk of cerebral ischemia, particularly during cocaine intoxication. Given that the PFC is a primary target for the neurovascular toxicity caused by cocaine, improving CBF could be particularly advantageous for the recovery of executive function and self-regulation. Both clinical and preclinical studies investigating chronic cocaine exposure have consistently demonstrated a decline in PFC function, which has been associated with compulsive-like cocaine intake in animals and relapse and cognitive impairments in humans (Büttner, 2011).

The chosen cocaine dosing regimens were designed to reflect different investigative focuses. The i.v. (1 mg/kg) was used as a challenge during imaging as it induces an acute reduction in cerebral blood flow, enabling observation of the temporal dynamics of vascular responses to cocaine. Meanwhile, to avoid invasive tail-vein catheterization for chronic cocaine treatment, i.p. administration (30 mg/kg daily for 14 days) was used to assess cocaine’s effect on long-term vascular dysfunction. Both doses are chosen based on prior in-vivo optical imaging studies (Du et al., 2021; Chen et al., 2016b) and also these doses result in cocaine plasma levels consistent to those observed in cocaine misusers (Morrell et al., 2011). Together, these two complementary cocaine treatment strategies strengthen the translational relevance of our findings by investigating both the immediate and persistent vascular effects of cocaine exposure at does that are clinically relevant.

One limitation of our study is that the measurements of vasoconstriction were taken while the animals were under isoflurane anesthesia. Isoflurane has the potential to counteract the vasoconstriction caused by cocaine by producing cerebral vasodilation, as shown in previous studies (Maekawa et al., 1986). Therefore, it is possible that the vasoconstrictive effects of cocaine were attenuated by the anesthesia. To mitigate this, we used minimal maintenance concentration (<2 %) and ensured that all experimental groups were imaged under similar anesthetic conditions to control systemic effects. In addition, we continuously monitored physiological parameters (e.g., heart rate, respiration) to maintain a similar baseline across animals. Furthermore, isoflurane anesthesia does not alter arterial carbon dioxide tension (PCO2) or coronary sinus oxygen tension (PO2), which otherwise would have confounded our findings with ΔHbO. While imaging in awake animals could reduce anesthesia related bias, motion and stress of the animals makes in-vivo imaging very challenging. Though, a mild, non-significant decrease in Δ[HbT] was observed following saline i.v. injection, potentially reflecting a transient physiological response to intravenous injection. Both the control and cocaine groups received identical volumes of i.v injections, which allowed control for this procedural factor. Another limitation is the translation of findings from an animal model to the human condition. In real life, cocaine misusers often use multiple drugs, which could also contribute to cocaine-associated stroke and TIA, whereas in our study, we only treated mice with cocaine. While the current study focused on imaging based assessments of neurovascular responses induced by cocaine, incorporating behavioral analysis would have enhanced the translational impact by linking physiological changes to functional outcomes. In future work, we will explore behavioral correlates of vascular dysfunction to provide a better understanding of cocaine’s effects on the brain. Finally, L-NMMA injection did not impair regional neurovascular coupling (White et al., 1999) and caused a 65 % decrease in NOS activity in humans (Stamler et al., 1994) and a 50 % reduction in rats (Iadecola et al., 1994). We observed significant improvement in cocaine-induced CBF reduction and vasoconstriction with L-NMMA pretreatment.

In summary, our study demonstrated significant vascular changes in the neurovascular trees within the PFC following chronic cocaine exposure. Comparison of the vascular changes between baseline (Day 0) and after chronic cocaine (Day 16) revealed enhanced vasoconstriction in WT whereas vasoconstriction was reduced in cocaine treated iNOS-KO and iNOS-Inh mice. Meanwhile, dynamic imaging showed cocaine induced hemodynamic changes were also reduced in iNOS-KO mice whereas microvascular density (e.g., capillary) was increased (also in iNOS-Inh mice). This indicates that angiogenesis might help mitigate cocaine’s vasoconstricting effects in CBF. These results suggest that strategies to inhibit iNOS could help reduce cerebral ischemia risk in individuals with cocaine use disorders.

Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.brainresbull.2025.111490.

Data availability

Data will be made available on request.