Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2021 Jan 1.
Published in final edited form as: Drug Alcohol Depend. 2019 Oct 18;206:107637. doi: 10.1016/j.drugalcdep.2019.107637

Acute cocaine administration alters permeability of blood-brain barrier in freely-moving rats— Evidence using miniaturized fluorescence microscopy

Jeffrey L Barr a, G Cristina Brailoiu b, Mary E Abood a, Scott M Rawls a, Ellen M Unterwald a, Eugen Brailoiu a
PMCID: PMC6980767  NIHMSID: NIHMS1544496  PMID: 31734036

Abstract

Background

Cocaine has a variety of negative effects on the central nervous system, including reports of decreased barrier function of brain microvascular endothelial cells. However, few studies have directly shown the effects of cocaine on blood-brain barrier (BBB) function in vivo. The miniature integrated fluorescence microscope (i.e., miniscope) technology was used to visualize cocaine-induced changes in BBB permeability in awake, freely-moving rats.

Methods

The miniscope was implanted in the prefrontal cortex of adult male rats. After recovery and acclimation, rats received an injection of cocaine (5–20 mg/kg ip) 15 minutes following iv infusion of sodium fluorescein, a low molecular weight tracer. Fluorescence intensity was recorded in vivo via the miniscope for 30 minutes or 24 hours post cocaine administration and served as an indicator of BBB permeability.

Results

Results demonstrate that cocaine increased the sodium fluorescein extravasation in brain microcirculation in a dose-dependent manner 30 minutes, but not 24 hours after administration.

Conclusion

We report for the first time using direct visualization of brain microcirculation with the miniscope technology in awake, freely-moving rats, that acute cocaine administration produced a transient increase in the BBB permeability.

Keywords: Cocaine, Blood-Brain Barrier, Sodium Fluorescein, Rat

1. Introduction

The blood-brain barrier (BBB) is a functional and structural interface that separates the circulation from the central nervous system and ensures the maintenance and regulation of the neural microenvironment (Hawkins and Davis, 2005). The BBB allows the transport of nutrients, removal of metabolites, and protection of the brain (Abbott et al., 2010). BBB permeability is mainly controlled by the monolayer of endothelial cells connected via tight and adherens junctions (Hawkins and Davis, 2005).

Cocaine remains a major drug of abuse, and reports indicate that cocaine can alter the BBB permeability via multiple mechanisms (Sharma et al., 2009). In brain microvascular endothelial cells cocaine, similarly to HIV, down-regulates tight junction proteins (Dhillon et al., 2008; Toborek et al., 2005). Cocaine alters expression of intracellular adhesion molecule 1, vascular cell adhesion molecule 1, endothelial-leukocyte adhesion molecule, and promotes neuro-inflammation (Fiala et al., 2005; Fiala et al., 1998; Kousik et al., 2012). Other studies indicate that cocaine acts via Sigma-1 receptors (Narayanan et al., 2011), modulates brain endothelial cell function by inhibiting store-operated calcium entry (Brailoiu et al., 2016; Rosado, 2016) and increases endothelial permeability (Yao et al., 2011). The effect of cocaine on the BBB contributes to potentiation of HIV-associated neurocognitive disorders (Cai et al., 2016; Fiala et al., 2005; Kousik et al., 2012). More recently, cocaine has been reported to impair the BBB permeability by increasing the expression and activity of prolidase that catalyzes collagen degradation during BBB remodeling (Ysrayl et al., 2019).

These studies provide important evidence that cocaine disrupts BBB function and suggest potential mechanisms involved. A limitation to these reports is that they were performed using in vitro cell systems. We used a miniature integrated fluorescence microscope (i.e., miniscope) to visualize in real-time cocaine-induced changes in BBB permeability in awake rats.

2. Materials and Methods

2.1. Animal Subjects

Adult male Sprague Dawley rats (Charles River Laboratories Inc.) were housed on a 12-hour light/dark cycle with free access to chow and water. Animal protocols were approved by the Institutional Animal Care and Use Committee of Temple University. The study followed the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines and the National Institutes of Health guide for the Care and Use of Laboratory Animals.

2.2. Chemicals

Cocaine was supplied by the National Institute on Drug Abuse’s Drug Supply Program. Cocaine was dissolved in saline and injected ip in a volume of 1 mL/kg body weight. Sodium fluorescein (Sigma–Aldrich, St. Louis, MO), was dissolved in saline and injected iv.

2.3. Surgical Procedures and Assessment of BBB Permeability

Surgical methods were as described previously (Barr et al., 2015). Briefly, rats were anesthetized with isoflurane, and imaging cannula (Miniscope GRIN lens, Doric Lenses, Inc., Quebec, Canada) implanted into the prefrontal cortex using the following stereotaxic coordinates from bregma (AP: 3mm, ML: 0.5 mm, DV: 2.6 mm) (Paxinos and Watson, 2013). After two weeks of recovery and habituation experiments began.

2.3.1. Experiment 1. Cocaine Dose-Response on BBB Permeability

Sodium fluorescein (Na-F; 200 mg/kg iv) was injected through the tail vein followed 15 min later by cocaine (5–20 mg/kg, ip) or saline. Na-F extravasation, as an indicator of BBB permeability, was measured immediately prior to (i.e., baseline) and again 3–30 min following saline or cocaine. Fluorescence intensity (Ex/Em 488/520 nm) was measured in 10 regions of interest (ROIs) in close proximity of microvessels. The same 10 ROIs were recorded prior to and following cocaine administration using a within-subjects design. Fluorescence was visualized, recorded and analysed post-acquisition using Doric Studio software (Doric Lenses, Inc).

2.3.2. Experiment 2. Long-Term Cocaine Effects on BBB

This study was performed to determine if the effects of cocaine on BBB permeability were present 24 hours after cocaine administration. Rats were injected with Na-F and fluorescence was measured with the miniscope 15 min later in order to obtain baseline measurements for 10 ROIs in close vicinity of the microvessels. Fluorescence was measured again 48 hours later when it was no longer detected. Cocaine (20 mg/kg, ip) or saline was administered, and 24 hours later, Na-F was injected and fluorescence (intensity of the same 10 ROIs) was assessed 15 min later.

2.4. Data Analysis

A within-subjects design was used wherein fluorescence intensity following saline or cocaine was compared with fluorescence intensity immediately prior to the injection (baseline), and reported as percent of baseline. For Experiment 1, two-way repeated measures ANOVA (with one factor repetition) was used to assess significant differences between treatment groups, followed by Tukey’s multiple comparisons post-hoc tests. A t-test was used to assess significance for Experiment 2.

3. Results

3.1. Cocaine Induces a Dose-Dependent Increase in Sodium Fluorescein Extravasation (Exp. 1)

Brain microcirculation in the prefrontal cortex was visualized with the miniscope following Na-F administration; Na-F extravasation was quantified as a measure of BBB permeability. Cocaine produced a time- and dose-dependent increase in Na-F extravasation as compared with saline administration (Fig. 1A; n=5 rats/dose). Two–way repeated measures ANOVA showed a significant effect of cocaine on Na-F extravasation (F(3, 16)=195.0, P<0.0001), a significant effect of time (F(7, 112)=265.3, P<0.0001) and a significant interaction (F(21, 112)=51.51, P<0.0001). Post-hoc analysis showed that at the cocaine 20 mg/kg dose, Na-F fluorescence was higher than that following saline (P<0.01), 5 mg/kg cocaine (P<0.01) and 10 mg/kg cocaine (P<0.05) at time points 5–30 min. Cocaine 10 mg/kg significantly increased Na-F extravasation at 10–30 min post injection as compared to saline and 5 mg/kg cocaine (P<0.01). Cocaine 5 mg/kg was not significantly different than saline (P>0.05) and there were no significant differences at 3 min post-cocaine. Examples of Na-F fluorescence immediately before (control baseline) and 30 min after injection of saline or cocaine (20 mg/kg) are shown in Fig. 1B. Pseudocolor images are shown in Fig. 1C. Cocaine increased Na-F extravasation, indicating leakage of Na-F from the vasculature and an increase in BBB permeability.

Figure 1. Cocaine induces a time- and dose-dependent increase in sodium fluorescein (Na-F) extravasation.

Figure 1.

Open in a new tab

A, Na-F extravasation is shown over time after injection of saline or cocaine (5, 10 and 20 mg/kg, ip). Cocaine (10 and 20 mg/kg) produced an increase in Na-F extravasation. Data are expressed as average fluorescence intensity (± SEM) for 10 RO1s, normalized to control baseline fluorescence measured immediately prior to injection of saline or cocaine in the same subject. (*) P<0.01 as compared with saline; (#) P<0.05 compared with cocaine 10 mg/kg or P<0.01 compared with cocaine 5 mg/kg. N=5 rats per treatment group. B, Examples of autofluorescence before Na-F injection and of Na-F fluorescence visualized with miniscope in rat prefrontal cortex before (control baseline) and 30 min after injection of saline (top) or cocaine (20 mg/kg, bottom). C, Pseudocolor images with calibration bar of images shown in panel B. Examples of 2 ROIs are indicated in panels B and C.

3.2. Cocaine Produces a Transient Increase in Sodium Fluorescein Extravasation (Exp. 2)

Baseline Na-F extravasation was quantified and, using a within-subjects design, miniscope imaging was repeated 48 hours later (Fig 2). As expected based on the clearance of Na-F, no fluorescence was detectable 48 hours later (Fig. 2). Cocaine (20 mg/kg ip) was administered, followed 24 hours later by Na-F and miniscope imaging repeated. Extravasation of Na-F 24 hours after cocaine administration was not significantly different from baseline Na-F extravasation (P=0.215; Fig. 2). This result indicates the transient effect of a single dose of cocaine on BBB permeability which lasts less than 24 hours.

Figure 2. Cocaine had no effect on sodium fluorescein (Na-F) extravasation 24 hours after administration.

Figure 2.

Open in a new tab

A, Summary of the experimental design. B, Comparison of Na-F extravasation in control (baseline Na-F) and 24 hours after a single injection of cocaine (20 mg/kg ip). Baseline Na-F extravasation was determined by averaging the fluorescence intensity for 10 ROIs in the vicinity of microvessels 15 min after iv administration of Na-F. Extravasation of Na-F 24 hours after cocaine was not significantly different (P = 0.215) from baseline Na-F extravasation. Data are expressed as average fluorescence intensity (± SEM) after normalization to control baseline fluorescence; N=5 rats. C, Examples of pseudocolor images with calibration bar.

4. Discussion

Changes in BBB permeability in response to several drugs and conditions have been characterized using in vitro models (Deli et al., 2005). In vivo studies of BBB permeability assess extravasation of endogenous tracers or injected molecules that cannot cross an intact BBB (Wunder et al., 2012). Evans Blue (MW 961 Da) and Na-F (MW 376 Da) are commonly used for this purpose (Yen et al., 2013), and their accumulation in the brain is assessed ex vivo (Brailoiu et al., 2018; Leo et al., 2019). In vivo two/multiphoton laser scanning microscopy allows the high-resolution dynamic imaging of BBB (Nishimura et al., 2006). While this technique permits time-lapse BBB imaging and correlation with neuronal responses, it is performed in anesthetized animals (Zhang and Murphy, 2007).

The miniscope is a transformative technology developed to assess neuronal function in vivo (Ghosh et al., 2011). An important advantage of examining the BBB permeability with the miniscope over classical methods is the higher anatomical selectivity, as fluorescent tracer extravasation is examined at selected ROIs in close proximity to the brain microvessels. Importantly, the miniscope technique allows the assessment of fluorescent tracer extravasation in real-time in freely-moving animals thus avoiding any potential effect of the anesthetic. Several anesthetic agents can impact BBB function in rodent models of neurotrauma (Sharma et al., 2019).

We assessed the effect of cocaine on the BBB permeability in vivo using miniscope, as previous reports suggest that cocaine may impair the barrier function (Sharma et al., 2009). We found a time- and dose-dependent increase in Na-F extravasation following acute cocaine administration, indicative of increased BBB permeability. Of note, Na-F fluorescence was undetectable when re-imaged 48 hours later which is in agreement with the reported pharmacokinetics of low molecular weight fluorescein-conjugated dextrans (Mehvar and Shepard, 1992). Na-F extravasation 24 hours after cocaine administration was not different from pre-cocaine baseline measurements. Previous studies indicate that cocaine is undetectable in the plasma 24 hours after injection (20 mg/kg) (Nayak et al., 1976). Since we identified that cocaine increased Na-F extravasation 5–30 minutes after administration but not 24 hour later, we conclude that the effect of a single injection of cocaine on BBB permeability is transient. It will be important in future studies to determine if chronic cocaine administration damages BBB function in a sustained manner.

5. Conclusions

Here we provide the first evidence that acute cocaine administration produces a transient increase in the BBB permeability in freely moving rats. Loss of barrier function may increase cocaine-induced neurotoxicity perhaps through enhanced cellular stress or edema, or by permitting increased entry of toxins or microbes into the CNS, supporting the need for further investigation of mechanisms involved in changes in BBB in the presence of cocaine.

Highlights.

  • The effect of cocaine on BBB was examined using miniscope implanted in adult rats.

  • Cocaine transiently increased Na fluorescein extravasation in brain microvessels.

  • In awake rats cocaine increased BBB permeability in dose-and time-dependent manner.

Acknowledgments

Role of Funding Source

This work was supported by the NIH grant P30DA013429 (to EMU).

Abbreviations

BBB

blood-brain barrier

miniscope

miniature integrated fluorescence microscope

Na-F

sodium fluorescein

ROIs

regions of interest

Footnotes

Conflict of Interest

No conflict declared.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  1. Abbott NJ, Patabendige AA, Dolman DE, Yusof SR, Begley DJ, 2010. Structure and function of the blood-brain barrier. Neurobiology of disease 37(1), 13–25. [DOI] [PubMed] [Google Scholar]
  2. Barr JL, Deliu E, Brailoiu GC, Zhao P, Yan G, Abood ME, Unterwald EM, Brailoiu E, 2015. Mechanisms of activation of nucleus accumbens neurons by cocaine via sigma-1 receptor-inositol 1,4,5-trisphosphate-transient receptor potential canonical channel pathways. Cell calcium 58(2), 196–207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brailoiu E, Barlow CL, Ramirez SH, Abood ME, Brailoiu GC, 2018. Effects of Platelet-Activating Factor on Brain Microvascular Endothelial Cells. Neuroscience 377, 105–113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brailoiu GC, Deliu E, Console-Bram LM, Soboloff J, Abood ME, Unterwald EM, Brailoiu E, 2016. Cocaine inhibits store-operated Ca2+ entry in brain microvascular endothelial cells: critical role for sigma-1 receptors. The Biochemical journal 473(1), 1–5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cai Y, Yang L, Callen S, Buch S, 2016. Multiple Faceted Roles of Cocaine in Potentiation of HAND. Current HIV research 14(5), 412–416. [DOI] [PubMed] [Google Scholar]
  6. Deli MA, Abraham CS, Kataoka Y, Niwa M, 2005. Permeability studies on in vitro blood-brain barrier models: physiology, pathology, and pharmacology. Cellular and molecular neurobiology 25(1), 59–127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dhillon NK, Peng F, Bokhari S, Callen S, Shin SH, Zhu X, Kim KJ, Buch SJ, 2008. Cocaine-mediated alteration in tight junction protein expression and modulation of CCL2/CCR2 axis across the blood-brain barrier: implications for HIV-dementia. Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology 3(1), 52–56. [DOI] [PubMed] [Google Scholar]
  8. Fiala M, Eshleman AJ, Cashman J, Lin J, Lossinsky AS, Suarez V, Yang W, Zhang J, Popik W, Singer E, Chiappelli F, Carro E, Weinand M, Witte M, Arthos J, 2005. Cocaine increases human immunodeficiency virus type 1 neuroinvasion through remodeling brain microvascular endothelial cells. Journal of neurovirology 11(3), 281–291. [DOI] [PubMed] [Google Scholar]
  9. Fiala M, Gan XH, Zhang L, House SD, Newton T, Graves MC, Shapshak P, Stins M, Kim KS, Witte M, Chang SL, 1998. Cocaine enhances monocyte migration across the blood-brain barrier. Cocaine’s connection to AIDS dementia and vasculitis? Advances in experimental medicine and biology 437, 199–205. [DOI] [PubMed] [Google Scholar]
  10. Ghosh KK, Burns LD, Cocker ED, Nimmerjahn A, Ziv Y, Gamal AE, Schnitzer MJ, 2011. Miniaturized integration of a fluorescence microscope. Nature methods 8(10), 871–878. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hawkins BT, Davis TP, 2005. The blood-brain barrier/neurovascular unit in health and disease. Pharmacological reviews 57(2), 173–185. [DOI] [PubMed] [Google Scholar]
  12. Kousik SM, Napier TC, Carvey PM, 2012. The effects of psychostimulant drugs on blood brain barrier function and neuroinflammation. Frontiers in pharmacology 3, 121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Leo LM, Familusi B, Hoang M, Smith R, Lindenau K, Sporici KT, Brailoiu E, Abood ME, Brailoiu GC, 2019. GPR55-mediated effects on brain microvascular endothelial cells and the blood-brain barrier. Neuroscience 414, 88–98. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Mehvar R, Shepard TL, 1992. Molecular-weight-dependent pharmacokinetics of fluorescein-labeled dextrans in rats. Journal of pharmaceutical sciences 81(9), 908–912. [DOI] [PubMed] [Google Scholar]
  15. Narayanan S, Mesangeau C, Poupaert JH, McCurdy CR, 2011. Sigma receptors and cocaine abuse. Current topics in medicinal chemistry 11(9), 1128–1150. [DOI] [PubMed] [Google Scholar]
  16. Nayak PK, Misra AL, Mule SJ, 1976. Physiological disposition and biotransformation of (3H) cocaine in acutely and chronically treated rats. The Journal of pharmacology and experimental therapeutics 196(3), 556–569. [PubMed] [Google Scholar]
  17. Nishimura N, Schaffer CB, Friedman B, Tsai PS, Lyden PD, Kleinfeld D, 2006. Targeted insult to subsurface cortical blood vessels using ultrashort laser pulses: three models of stroke. Nature methods 3(2), 99–108. [DOI] [PubMed] [Google Scholar]
  18. Paxinos G, Watson C, 2013. The Rat Brain In Stereotaxic Coordinates, Seventh Edition. Academic Press. [Google Scholar]
  19. Rosado JA, 2016. Sigma-1 receptors: a new pathway for the modulation of store-operated calcium entry. The Biochemical journal 473(3), e9–e10. [DOI] [PubMed] [Google Scholar]
  20. Sharma HS, Muresanu D, Sharma A, Patnaik R, 2009. Cocaine-induced breakdown of the blood-brain barrier and neurotoxicity. International review of neurobiology 88, 297–334. [DOI] [PubMed] [Google Scholar]
  21. Sharma HS, Muresanu DF, Nozari A, Castellani RJ, Dey PK, Wiklund L, Sharma A, 2019. Anesthetics influence concussive head injury induced blood-brain barrier breakdown, brain edema formation, cerebral blood flow, serotonin levels, brain pathology and functional outcome. International review of neurobiology 146, 45–81. [DOI] [PubMed] [Google Scholar]
  22. Toborek M, Lee YW, Flora G, Pu H, Andras IE, Wylegala E, Hennig B, Nath A, 2005. Mechanisms of the blood-brain barrier disruption in HIV-1 infection. Cellular and molecular neurobiology 25(1), 181–199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wunder A, Schoknecht K, Stanimirovic DB, Prager O, Chassidim Y, 2012. Imaging blood-brain barrier dysfunction in animal disease models. Epilepsia 53 Suppl 6, 14–21. [DOI] [PubMed] [Google Scholar]
  24. Yao H, Duan M, Buch S, 2011. Cocaine-mediated induction of platelet-derived growth factor: implication for increased vascular permeability. Blood 117(8), 2538–2547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Yen LF, Wei VC, Kuo EY, Lai TW, 2013. Distinct patterns of cerebral extravasation by Evans blue and sodium fluorescein in rats. PloS one 8(7), e68595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ysrayl BB, Balasubramaniam M, Albert I, Villalta F, Pandhare J, Dash C, 2019. A Novel Role of Prolidase in Cocaine-Mediated Breach in the Barrier of Brain Microvascular Endothelial Cells. Scientific reports 9(1), 2567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Zhang S, Murphy TH, 2007. Imaging the impact of cortical microcirculation on synaptic structure and sensory-evoked hemodynamic responses in vivo. PLoS biology 5(5), e119. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES