Closed cranial window animal preparation. Animal handling and care followed the NIH Guide for Care and Use of Laboratory Animals. All protocols were approved by the La Jolla Bioengineering Institutional Animal Care and Use Committee. 8 to 10-week old C57Bl/6J and Balb/cJ mice (Jackson Laboratories, ME) were implanted with a closed cranial window model as described elsewhere16. Briefly, mice were anesthetized with ketamine-xylazine and were administered dexamethasone (0.2mg/Kg), carprofen (5mg/Kg) and ampicillin (6mg/kg) subcutaneously, to prevent post-surgical swelling of the brain, inflammatory response, and infection. After shaving the head and cleansing with ethanol 70% and betadine, the mouse was placed on a stereotaxic frame and the head immobilized using ear bars. The scalp was removed with sterilized surgical instruments and lidocaine-epinephrine was applied on the periosteum, which was then retracted to expose the skull. A 3-4mm diameter skull opening was made in the left parietal bone using a surgical drill. Under a drop of saline, the craniotomy was lifted away from the skull with very thin-tip forceps and gelfoam previously soaked in saline was applied to the dura mater to stop any eventual small bleeding. The exposed area was covered with a 5mm glass cover slip secured with cyanocrylate-based glue and dental acrylic. Carprofen and ampicillin were given daily for 3–5 days after recovery from surgery. Mice presenting signs of pain or discomfort were euthanized with 100mg/kg of euthasol IP. Two to three weeks after surgery, mice fulfilling the inclusion criteria (see below) were inoculated with P. berghei ANKA and, on day 6 of infection, they were lightly anesthetized with isoflurane (4% for induction, 1–2% for maintenance) and held on a stereotaxic frame for measurements of pH and pO2.
Inclusion criteria. Animals were suitable for the experiments if: 1) animal behavior was normal and 2) microscopic (x350 magnification) examination of the cranial window did not reveal signs of edema or bleeding.
Parasite infection. Animals were inoculated with an IP injection of 1 x 106 Plasmodium berghei ANKA parasites expressing the green fluorescent protein (PbA-GFP, a donation from the Malaria Research and Reference Reagent Resource Center – MR4, Manassas, VA; deposited by CJ Janse and AP Waters; MR4 number: MRA-865). Parasitemia, body weight, rectal temperature and clinical status (using six simple tests adapted from the SHIRPA protocol, as previously described) were monitored daily from day 4 of the infection17. Parasitemia was checked using flow cytometry by detecting the number of fluorescent GFP-expressing pRBCs in relation to 10,000 RBCs. ECM was diagnosed when one or more of the following clinical signs of neurological involvement were observed: ataxia, limb paralysis, poor righting reflex, seizures, roll-over, or coma.
Physiological ranges of the variables measured for the animal species used. Two groups of animals, C57BL/6 (n = 6) and BALB/c (n = 6), instrumented with the closed cranial window were used to characterize normal microhemodynamic (vessel diameter and blood flow), intravascular and perivascular PO2s and pH in the pial microenvironment.
Experimental Groups. Group 1 aimed to establish the effects of PbA infection in microhemodynamics, intravascular and perivascular PO2s and pH in the pial microenvironment. The group consisted of ECM-susceptible C57BL/6 (Infected, n = 14) and ECM-resistant BALB/c (Infected, n = 9) mice. Uninfected C57BL/6 (n = 6) and BALB/c (n = 6) mice were included as controls. Control animals were manipulated in the exact same way as the infected mice (except for the infection itself). C57BL/6 ECM animals at day 6 of infection were divided in two cohorts: Early-stage ECM, presenting mild to moderate drops in body temperature (> 34 < 36°C) and Late-stage ECM, showing marked drops in body temperature (< 33°C). Another group, Group 2, was included to establish the relation between vascular inflammation resulting from PbA infection and microhemodynamics and oxygenation in relation to ECM pathophysiological changes. The group consisted of C57BL/6 (Infected, n = 9) mice to which leukocyte adhesion, blood flow and PO2 levels were measured. Similarly, as in Group 1, the ECM animals at day 6 of infection were divided in two cohorts: Early-stage ECM and Late-stage ECM. All experiments were repeated at least once.
Experimental Setup. Animals were lightly anesthetized with isoflurane (4% for induction, 1–2% for maintenance). They were secured to the microscopic stage of an intravital microscope (BX51WI, Olympus, New Hyde Park, NY) on a stereotaxic frame with the head gently held with ear bars for epi-illumination imaging. Body temperature, measured pre-anesthesia, was maintained with a heating pad. The tissue image was projected onto a charge-coupled device camera (COHU 4815) connected to a videocassette recorder and viewed on a monitor. Measurements were carried out using a 40X (LUMPFL-WIR, numerical aperture 0.8, Olympus) water immersion objective. The animals did not recover from anesthesia, as they were euthanized (Euthasol 100mg/kg, IP) right after the intravital microscopy measurements.
Microhemodynamics. A video image-shearing method was used to measure vessel diameter (D)18. Changes in arteriolar and venular diameter from baseline were used as indicators of a change in vascular tone. Arteriolar and venular centerline velocities were measured on-line using the photodiode cross-correlation method (Photo Diode/Velocity Tracker Model 102B, Vista Electronics, San Diego, CA). The measured centerline velocity (V) was corrected according to vessel size to obtain the mean RBC velocity19,20. Blood flow (Q) was calculated from the measured values as Q = π × V (D/2)^2. This calculation assumes a parabolic velocity profile and has been found to be applicable to tubes of 15–80 µm internal diameters and for Hcts in the range of 6–60%19,20.
Microvascular PO2 distribution. High resolution non-invasive microvascular pO2 measurements were made using phosphorescence quenching microscopy (PQM)21. PQM is based on the relationship between the decay rate of excited Palladium-mesotetra-(4-carboxyphenyl) porphyrin (Frontier Scientific Porphyrin Products, Logan, UT) bound to albumin and the O2 concentration according to the Stern-Volmer Eq. 21,22. The method was used previously in microcirculatory studies to determine pO2 levels in different tissues22. pO2. measurements by PQM were obtained following these steps for all groups: 1) the probe was injected (tail injection of 15 mg/kg at a concentration of 10 mg/ml of the phosphorescence complex 10 min before O2 measurements); 2) the tissue was illuminated (pulsed light at 420 nm wavelength) to excite the probe into its triplet state; 3) the emitted phosphorescence (680 nm wavelength) was collected and analyzed to yield the phosphorescence lifetime; and 4) the phosphorescence lifetime was converted into O2 concentration, pO2.The phosphorescence lifetimes are concentration independent, which permit extravascular fluid pO2 measurements, although the dye albumin complex that extravasates is very small. Extravascular fluid pO2 was measured in regions in between functional capillaries. PQM allows for precise localization of the pO2 measurements without subjecting the tissue to injury. These measurements provide a detailed understanding of microvascular O2 distribution and indicate whether O2 is delivered to the interstitial areas.
Hematocrit and hemoglobin. Blood was collected from the tail in heparinized glass capillaries. Hemoglobin was determined spectrophotometrically from a single drop of blood in a B-Hemoglobin analyzer (Hemocue, Stockholm, Sweden). Hematocrit was estimated by centrifugation.
Oxygen delivery and extraction. Oxygen delivery, DO2 was approximated as
$$D{O}_{2}=\left[(RB{C}_{Hb}\times \gamma \times {S}_{A})\right]\times {Q}_{A}$$
1
where RBCHb is the total Hb (g/dL), \(\gamma\) is the O2 carrying capacity of saturated Hb, approximated as 1.34 mL O2/g Hb, SA is arteriolar blood oxygen saturation, and QA is arteriolar flow. Similarly, arterio-venous oxygen extraction (O2 A-V Extraction, or VO2) was approximated as
$$DV=\left[(RB{C}_{Hb}\times \gamma \times {S}_{A-V})\right]\times {Q}_{A-V}$$
2
where SA−V is the difference between arteriolar and venular oxygen saturation and QA−V is the average of arteriolar and venular flow rate. O2 saturations were approximated using the blood O2 equilibrium curve.
Statistical Analysis. Results are presented as mean ± standard deviation. Data between groups was analyzed using a non-parametric Kruskal-Wallis test. When appropriate, post hoc analyses were performed with the Dunns’ multiple comparison test. Microvascular O2 content and O2 delivery were compared using a general linear mixed model as a function of either diameter or flow. Data were linearized by log-log transformation to allow for analysis. Differences in radial and axial O2 fluxes were compared using an analysis of covariance (ANCOVA) from the aforementioned general linear mixed model. All statistics were calculated using GraphPad Prism 9.1.2. Changes were considered statistically significant if p < 0.05.