The BBB is a membranous structure with the primary role of protecting the CNS from potentially harmful substances found in blood, while simultaneously allowing the passage of substances needed for physiologic functions. This fundamental concept of the function and role of the BBB has not essentially changed since 1885, when Ehrlich1 noticed that the BBB has specialized properties that enable the selective passage of only certain circulating factors. However, the BBB is not a rigid structure but rather a dynamic interface with a range of interrelated functions; this dynamic interface thereby generates the physical, transport, enzymatic, and immune regulatory functions.2
The BBB has been described in all vertebrates; however, its structure and physiologic function have been investigated mainly in mammals and are poorly characterized in lower vertebrates. For most vertebrates as well as teleost fish, the composition and function of the BBB are coordinated by interactions between neurons, astrocytes, and endothelial cells. Transcellular tracts use apical and basolateral transporters and canals associated with the membranes that conduct substances through the epithelial cells. Paracellular permeability is regulated by the selection of molecules on the basis of charge and size. Paracellular transit of substances is primarily controlled by the tight junction complex located between epithelial cells on the most apical region of the lateral cellular membrane.3
Most experiments conducted to investigate the influence of NO on the BBB permeability in mammals have suggested that NO increases the permeability of the BBB, which enables the passage of substances into the brain by passive diffusion.4,5 In situ perfusion studies6,7 on rats revealed that NO mediates opening of the BBB to a certain extent, depending on NO concentration and the type of NO donor. The precise molecular mechanism for increased BBB permeability is complex and has not been completely clarified.8 However, alterations in BBB permeability might be associated with peroxynitrite formation8–10 as well as with NO-mediated activation of various signaling pathways, which can lead to inflammation and possibly to the development of vasogenic edema and secondary brain damage.9,11,12
Nitric oxide is characterized as an important biological regulator and therefore has become a fundamental component in the fields of neuroscience, physiology, and immunology. Thus, there has been a substantial increase in investigating its role and function in various animal species during the past 20 years.13 Because of the short half-life of NO in vivo, NO production in experimental conditions is primarily estimated by determining the concentrations of nitrite (NO2−) and nitrate (NO3−) end products.14 Measurement of the nitrite concentration, nitrate concentration, or a combination of the nitrite and nitrate concentration is routinely used as an index of NO production.15
The presence of NO has been confirmed in the CNS of all investigated vertebrates, from lampreys to mammals, and the role of NO differs among various animal species.16 During investigation of the effects of NO in the CNS of teleost fish, the greatest attention has been given to experiments related to the effect of hypercapnia or anoxia on circulation in the brain. To our knowledge, the effect of NO on BBB permeability in lower vertebrates has not been evaluated. From a toxicologic point of view, taking into account that an intact BBB is important for protecting the brain, it is important to investigate clinical conditions that can affect the BBB's protection properties.17 Therefore, the purpose of the study reported here was to determine the effect of the NO donor glyceryl trinitrate on BBB permeability and subsequent consequences on brain anatomy in common carp (Cyprinus carpio L.).
Materials and Methods
Animals
The study was conducted with 148 healthy 2-year-old common carp that originated from a fish farm in Croatia. Body weight of fish ranged from 500 to 1,000 g. All fish were deemed healthy on the basis of results of a health examination. Some fish were used in ≥ 1 experiment. The study protocol was reviewed and approved by the Ethical Committee of the Faculty of Veterinary Medicine, University of Zagreb; Ethical Committee of the School of Medicine, University of Zagreb; and Ethical Committee of the Ministry of Science, Croatia.
Procedures
After the health examination was completed, fish were transferred to a laboratory of the Department for Biology and Pathology of Fish and Bees at the Faculty of Veterinary Medicine of the University of Zagreb. The laboratory used a constant flow-through system supplied by dechlorinated tap water. Sodium thiosulfate pentahydratea was used to dechlorinate water in the main storage tank. Dechlorinated water flowed from the storage tank into glass aquaria (capacity, 100 L/aquarium). Total volume of the system was approximately 6,000 L.
Fish were distributed among 37 aquaria (4 fish/aquarium) and allowed a 4-week adaptation period. During the adaptation period, fish were fed a commercial feed for carpb twice per day (1% of biomass/d). Water quality in the system was monitored daily. The pH and dissolved oxygen concentration were determined with a probe,c and total ammonia concentration was determined with a handheld colorimeter.d Water temperature in the system was between 16.1° and 17.5°C, pH was between 8.2 and 8.4, dissolved oxygen concentration was between 6.2 and 8.1 mg/L, and total ammonia concentration was between 0.09 and 0.15 μg/mL
Fish were anesthetized and euthanized in a separate tank by immersion in a solution of tricaine mesylate (MS-222).e A concentration of 30 mg/L was used to induce anesthesia (exposure time, 5 minutes), and euthanasia was performed with a concentration of 310 mg/L (exposure time, 3 minutes).
Determination of total serum nitrate and nitrite concentration, serum nitrate concentration, and serum nitrite concentration
Glyceryl trinitratef (1 mg/kg) was injected IP into 108 arbitrarily selected fish. A blood sample (1 mL) was collected from the caudal vessel of different treated fish 0.25 (n = 15 fish), 1 (16), 3 (15), 6 (18), 8 (15), 12 (12), and 24 (17) hours after injection. A blood sample was simultaneously collected from 7 untreated (control) fish. Blood samples were centrifuged (10 minutes at 1,500 × g at 4°C), and serum was filtered through a 10-kDa filter.g Samples were stored in a freezer at −20°C until analysis. A commercial test packageh was used to determine serum concentrations of nitrite and nitrate. Each sample was measured in triplicate. Concentrations were measured in a 2-step process in accordance with the manufacturer's instructions. The first step was conversion of nitrate to nitrite by use of nitrate reductase. The second step was the addition of Gries reagents, which converted nitrite into a deep purple azo compound. Photometric measurement of the absorbance attributable to this azo chromophore was used to determine the nitrite concentration. Total nitrate and nitrite concentration and the nitrite concentration were determined directly by use of the colorimetric assay, whereas nitrate concentration was derived indirectly by calculation.
Macroscopic and microscopic determination of BBB permeability by use of Evans blue dye
This experiment was conducted with 32 carp. Some of the fish had been used previously in experiments, and the interval between the preceding experiment and this experiment was 7 days. Carp were allocated to 4 groups (8 fish/group). Glyceryl trinitrate (1 mg/kg) was injected IP into 24 fish (3 groups); concurrently, sterile saline (0.63% NaCl) solution (physiologic saline solution for fish) was administered IP to the 8 fish of the control group. Fish injected with glycerol trinitrate were euthanized 6, 12, and 24 hours after injection (1 group/time point); control fish were euthanized 6, 12, and 24 hours after injection of saline solution.
For euthanasia, fish were anesthetized with MS-222, and then 2% Evans blue dye solutione (2 mg/kg) was administered IP, in accordance with a method described elswhere.18,19 A 30-minute period was allowed for solution circulation, and fish were then euthanized with MS-222. The pericardial cavity of each fish was surgically opened, and the bulbus arteriosus was perfused with 20 mL of saline solution followed by 20 mL of 4% formaldehyde to remove intravascular dye. The brain of each fish was carefully removed by means of excochleation and fixed in 4% formaldehyde.20
Fixed samples of carp brain were photographed. Staining of the individual parts of the CNS (front brain [telencephalon], interbrain [diencephalon], midbrain [mesencephalon], cerebellum [metencephalon], and spinal cord [medulla spinalis]) with Evans blue dye was graded on a scale from 0 to 3 (0 = areas with no stain, 1 = areas with slight amounts of stain, 2 = areas with moderate amounts of stain, and 3 = areas with intense amounts of stain).
Fixed samples of carp brain were embedded in paraffin and then cut with a microtome into transverse sections at a thickness of 5 μm. Eight sections were arbitrary selected from each brain sample and analyzed with fluorescent microscopy. The excitation wavelength maximum for Evans blue dye is between 470 and 540 nm, and emission wavelength is 680 nm.21 Leakage of Evans blue dye as a marker of increased BBB permeability appeared as a pericapillary halo (ie, intense red staining of the pericapillary space).
Determination of brain edema by use of the wet-dry weight method
In this experiment, carp were allocated to 3 groups (15 fish/group). Some of these fish had been used in a previous experiment; thus, there was a 1-week period between the preceding experiment and this experiment. Glyceryl trinitrate (1 mg/kg) was injected IP into fish of 2 groups, and saline solution was injected IP into fish of the third (control) group. Fish injected with glycerol trinitrate were euthanized 6 and 12 hours after injection (1 group at each time point), and control fish were euthanized 12 hours after injection of saline solution. The brain was removed from each euthanized fish and weighed to determine weight of the wet-matter content. Brains then were placed in a dry sterilizer for 24 hours at 105°C, after which they were again weighed to determine weight of the dry-matter content. Percentage of water in each brain was calculated by use of the following equation22: proportion of water in the brain = (wet-matter weight − [dry-matter weight/wet-matter weight]) × 100.
Determination of brain edema and vasodilation by use of H&E-stained brain preparations
This experiment involved 33 carp. Some of these fish had been used in a previous experiment; thus, there was a 1-week period between the preceding experiment and this experiment. Fish were allocated to 3 groups. Fish of 2 groups (10 fish/group) received an IP injection of glyceryl trinitrate (1 mg/kg) and were euthanized 6 and 24 hours after injection (1 group at each time point). Fish of the control group (n = 13) received an IP injection of saline solution and were euthanized 24 hours after injection. The brain was removed from each euthanized fish and fixed in 4% formaldehyde. Fixed brain tissue was embedded in paraffin, cut with a microtome into transverse 5-μm-thick sections, and stained with H&E dye. Changes characteristic of brain edema and vasodilation were analyzed with light microscopy. Brain edema was histopathologically diagnosed on the basis of generally accepted morphological criteria for cerebral edema (pallor of myelin, distention of perivascular and pericellular spaces, a loose or sieve-like appearance of myelinated areas, rarefaction of subpial spaces, a vacuolar appearance of the gray matter neuropil, and pools of protein-rich fluid).23 Vasodilation was diagnosed on the basis of histopathologic findings of dilated blood vessels filled with blood and thin, elongated, but regular endothelial cells. All slides were evaluated by the same investigator (EG) to ensure consistency.
Data analysis
Data were analyzed with a standard statistical computer program.i The Kolmogorov-Smirnov test was used to test the assumption of a normal distribution for all quantitative data prior to further analysis. Quantitative data were expressed as sample mean and SEM if normally distributed and as median and interquartile range if not normally distributed. Categorical variables were expressed as numbers and percentages. Values of P ≤ 0.05 were considered significant.
The t test and Mann-Whitney U test were used to test differences between groups for parametric and nonparametric data, respectively. Differences between multiple groups were tested with an ANOVA for parametric data and with the Kruskal-Wallis test for nonparametric data.
Differences in total nitrate and nitrite concentration, nitrite concentration, and nitrate concentration were analyzed with parametric tests. For analysis of differences among groups, an ANOVA and Tukey post-tests of multiple comparison were used.
The amount of Evans blue dye detected macroscopically in certain parts of the brain was evaluated with the Mann-Whitney U test. Differences in the percentage of water in the carp brain were evaluated with the Mann-Whitney test for independent samples. Differences in the percentage of water in the carp brain among groups were evaluated with the nonparametric Kruskal-Wallis test.
Results of microscopic analysis for leakage of Evans blue dye (assessed with fluorescent microscopy) and determination of brain edema and vasodilation on H&E preparations (assessed with light microscopy) were not statistically analyzed. Descriptive data were reported for those findings.
Results
Total serum nitrate and nitrite concentration, serum nitrate concentration, and serum nitrite concentration
Mean ± SEM total serum nitrate and nitrite concentration for the control group was 109.00 ± 2.67 μmol/L. Total serum nitrate and nitrite concentration was significantly higher, compared with the concentration for the control fish, for fish evaluated 0.25 (188.10 ± 10.65 μmol/L), 1 (173.70 ± 7.27 μmol/L), 6 (236.40 ± 28.92 μmol/L), 8 (255.20 ± 11.54 μmol/L), 12 (178.20 ± 7.45 μmol/L), and 24 (148.60 ± 3.41 μmol/L) hours after the injection of glyceryl trinitrate. No significant difference was detected between the concentration for the control group and that for fish evaluated 3 hours after injection of glyceryl trinitrate (127.50 ± 8.43 μmol/L).
Total serum nitrate and nitrite concentration for the group evaluated 0.25 hours after glyceryl trinitrate injection was significantly higher than that for the group evaluated 3 hours after injection and significantly lower than that for the group evaluated 8 hours after injection. Total serum nitrate and nitrite concentration for the group evaluated 1 hour after injection of glyceryl trinitrate was significantly higher than that for the group evaluated 3 hours after injection and significantly lower than that for the groups evaluated 6 and 8 hours after injection. Total serum nitrate and nitrite concentration was lowest for the group evaluated 3 hours after glyceryl trinitrate injection, which was significantly lower than the concentration for groups evaluated 6, 8, and 12 hours after injection. Groups evaluated 6 and 8 hours after injection of glyceryl trinitrate had a significantly higher total serum nitrate and nitrite concentration, compared with the concentration for groups evaluated 12 and 24 hours after injection.
A mild increase in the total serum nitrate and nitrite concentration was detected for the group evaluated 0.25 hours after glyceryl trinitrate injection. Total serum nitrate and nitrite concentration for the groups evaluated 1, 3, 12, and 24 hours after injection was similar to the value for the control group (Figure 1).
Mean ± SEM total serum nitrate and nitrite concentration in samples obtained from common carp (Cyprinus carpio L.) that were not treated (control group [Con]; n = 7) or from carp 0.25 (15), 1 (16), 3 (15), 6 (18), 8 (15), 12 (12), and 24 (17) hours after IP injection of glyceryl trinitrate (1 mg/kg). *Value differs significantly (P ≤ 0.05) from the value for the control group. †Value differs significantly (P ≤ 0.05) from the value for the group evaluated at 3 hours. ‡Value differs significantly (P ≤ 0.05) from the value for the group evaluated at 8 hours. § Value differs significantly (P ≤ 0.05) from the value for the group evaluated at 6 hours.
Citation: American Journal of Veterinary Research 76, 7; 10.2460/ajvr.76.7.615
The pattern for serum nitrate concentration matched that for the total serum nitrate and nitrite concentration. Mean ± SEM serum nitrate concentration for the control group was 99.20 ± 3.02 μmol/L. Mean serum nitrate concentration for the groups evaluated 0.25 (180.20 ± 11.52 μmol/L), 1 (168.20 ± 7.34 μmol/L), 6 (232.40 ± 28.55 μmol/L), 8 (236.90 ± 39.86 μmol/L), 12 (166.80 ± 6.71 μmol/L), and 24 (139.70 ± 4.87 μmol/L) hours after injection of glyceryl trinitrate was significantly higher, compared with the serum nitrate concentration for the control group. The serum nitrate concentration for the group evaluated 3 hours after injection of glyceryl trinitrate (113.20 ± 7.41 μmol/L) was similar to that for the control group.
The serum nitrate concentration for the group evaluated 0.25 hours after injection of glyceryl trinitrate was significantly higher than that for the group evaluated 3 hours after injection and significantly lower than that for the group evaluated 8 hours after injection. The serum nitrate concentration for the group evaluated 1 hour after injection with glyceryl trinitrate was significantly higher than that for the group evaluated 3 hours after injection and significantly lower than that for the groups evaluated 6 and 8 hours after injection. The serum nitrate concentration for the group evaluated 3 hours after injection of glyceryl trinitrate was significantly lower than the serum nitrate concentration for the groups evaluated 6, 8, and 12 hours after injection. Serum nitrate concentration for the groups evaluated 6 and 8 hours after injection of glyceryl trinitrate was significantly higher, compared with the serum nitrate concentration for the groups evaluated 12 and 24 hours after injection.
Mean ± SEM serum nitrite concentration for the control group (9.82 ± 3.02 μmol/L) did not differ significantly from the concentration for fish evaluated 0.25 (7.95 ± 2.92 μmol/L), 1 (5.52 ± 0.78 μmol/L), 3 (14.28 ± 2.49 μmmol/L), 6 (4.01 ± 1.01 μmol/L), 8 (16.17 ± 2.97 μmol/L), 12 (3.61 ± 0.77 μmol/L), and 24 (5.55 ± 0.85 μmol/L) hours after injection of glyceryl trinitrate. The nitrite concentration for the group evaluated 1 hour after injection of glyceryl trinitrate was significantly lower than the concentration for the groups evaluated 3 and 8 hours after injection. Serum nitrite concentration for fish evaluated 3 hours after injection of glyceryl trinitrate was significantly higher, compared with the serum nitrite concentration for the groups evaluated 6, 12, and 24 hours after injection. Serum nitrite concentration for fish evaluated 6 hours after injection of glyceryl trinitrate was significantly lower than that for the group evaluated 8 hours after injection. Fish evaluated 8 hours after injection with glyceryl trinitrate had the highest mean serum nitrite concentration, which was significantly higher than that for the groups evaluated 12 and 24 hours after injection of glyceryl trinitrate (Figure 2).
Mean ± SEM serum nitrite concentration in the carp of Figure 1. *Value differs significantly (P ≤ 0.05) from the value for the group evaluated at 3 hours. † Value differs significantly (P ≤ 0.05) from the value for the group evaluated at 8 hours. ‡ Value differs significantly (P ≤ 0.05) from the value for the group evaluated at 1 hour. § Value differs significantly (P ≤ 0.05) from the value for the group evaluated at 6 hours.
Citation: American Journal of Veterinary Research 76, 7; 10.2460/ajvr.76.7.615
The serum nitrite concentration was significantly lower than the serum nitrate concentration for all experimental groups. The pattern of changes in serum nitrite concentration over time was different from that of the total serum nitrate and nitrite concentration as well as from that of the serum nitrate concentration.
The percentage of nitrate in the total serum nitrate and nitrite concentration was higher than the percentage of nitrite for the control group (Table 1). Similarly, for all glyceryl trinitrate-treated groups, the percentage of nitrate (89.1% to 96.8%) in the total serum nitrate and nitrite concentration was higher than the percentage of nitrite (3.2% to 10.9%).
Percentages of nitrate and nitrite in the total serum nitrate and nitrite concentration for samples obtained from common carp (Cyprinus carpio L.) that were not treated (control group; n = 7) or from carp 0.25 (15), 1 (16), 3 (15), 6 (18), 8 (15), 12 (12), and 24 (17) hours after IP injection of glyceryl trinitrate (1 mg/kg).
| Group | Nitrate (%) | MACNitrite (%) (%) |
|---|---|---|
| Control | ||
| 0.25 h | ||
| 1 h | ||
| 3 h | ||
| 6 h | ||
| 8 h | ||
| 12 h | ||
| 24 h |
BBB permeability determined by use of Evans blue dye
Four carp (2 from the control group, 1 from the group euthanized 6 hours after injection of glyceryl trinitrate, and 1 from the group euthanized 12 hours after injection of glyceryl trinitrate) were excluded from the study because the procedure for dye administration was considered unsuccessful. These fish were not macroscopically or histologically analyzed because an error in dye administration was considered to have resulted in dye malabsorption.
Intensity of staining with Evans blue dye for certain brain areas of the control group and groups injected with glyceryl trinitrate and euthanized 6, 12, and 24 hours after injection were summarized (Table 2). All parts of the CNS (telencephalon, diencephalon, mesencephalon, metencephalon, and spinal cord) for the group euthanized 6 hours after injection of glyceryl trinitrate had a significantly higher degree of staining intensity, compared with the staining intensity of the control group and groups euthanized 12 and 24 hours after injection. Results indicated that the BBB was again impermeable by 12 and 24 hours after injection of glyceryl trinitrate. Mild staining of certain parts of the CNS (telencephalon, diencephalon, and spinal cord) for the control group of fish indicated a permeable BBB or leakage of dye through the area of the circumventricular organs (areas of the brain that lack a BBB).
Intensity of staining with Evans blue dye in various parts of the CNS obtained from carp* injected IP with sterile saline (0.63% NaCl) solution (control group) or injected IP with glyceryl trinitrate (1 mg/kg) and euthanized 6, 12, and 24 hours after injection.
| Glyceryl trinitrate | ||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Control (n = 6) | 6 hours (n = 7) | 12 hours (n = 7) | 24 hours (n = 8) | |||||||||||||
| Brain area | 0 | 1 | 2 | 3 | 0 | 1 | 2 | 3 | 0 | 1 | 2 | 3 | 0 | 1 | 2 | 3 |
| Telencephalon | 5 | 1 | 0 | 0 | 0 | 2 | 1 | 4 | 5 | 2 | 0 | 0 | 7 | 1 | 0 | 0 |
| Diencephalon | 3 | 3 | 0 | 0 | 0 | 0 | 0 | 7 | 2 | 4 | 1 | 0 | 1 | 7 | 0 | 0 |
| Mesencephalon | 6 | 0 | 0 | 0 | 0 | 3 | 4 | 0 | 7 | 0 | 0 | 0 | 7 | 1 | 0 | 0 |
| Metencephalon | 6 | 0 | 0 | 0 | 0 | 1 | 4 | 2 | 6 | 1 | 0 | 0 | 8 | 0 | 0 | 0 |
| Spinal cord | 4 | 2 | 0 | 0 | 0 | 0 | 0 | 7 | 2 | 5 | 0 | 0 | 5 | 3 | 0 | 0 |
Staining intensity was graded on a scale from 0 to 3 (0 = areas with no stain, 1 = areas with slight amounts of stain, 2 = areas with moderate amounts of stain, and 3 = areas with intense amounts of stain).
Each group initially comprised 8 carp; however, 4 carp (2 from the control group, 1 from the group euthanized 6 hours after injection of glyceryl trinitrate, and 1 from the group euthanized 12 hours after injection of glyceryl trinitrate) were excluded from the study because the procedure for dye administration was considered unsuccessful.
Histologic evaluation of thin sections of carp brain (3 preparations/brain sample) stained with Evans blue dye in vivo 6 hours after injection of glyceryl trinitrate revealed that there were signs of dye leakage into the pericapillary space, which suggested an increase in BBB permeability (Figure 3). Excluding areas of the circumventricular organs, no notable dye leakage was observed in the brain preparations of the control carp and carp injected with glyceryl trinitrate 12 and 24 hours before euthanasia.
Photomicrographs obtained by use of a fluorescent microscope of leakage of Evans blue dye through the BBB in thin sections of the cerebellum of the brains of representative carp. A—Leakage of albumin from the capillaries 6 hours after administration of glyceryl trinitrate (1 mg/kg) is evident as red staining. The large circle indicates large confluent areas of labeled blood-borne fluorophore (ie, Evans blue dye), and the small circle indicates a less affected part of the brain. B—In the fish from the control group (injected with saline [0.63% NaCl] solution), red fluorescence of Evans blue dye is visible only within the capillaries. Evans blue dye stain; bar = 800 μm for panel A and 200 μm for panel B.
Citation: American Journal of Veterinary Research 76, 7; 10.2460/ajvr.76.7.615
Brain edema determined by use of the wet-dry weight method
For the control group, mean percentage of brain wet matter was 74.2% (range, 72.2% to 76.1%). For the groups injected with glyceryl trinitrate and euthanized 6 and 24 hours after injection, mean percentage of brain wet matter was 78.1% (range, 77.0% to 80.2%) and 78.5% (range, 77.5% to 80.4%), respectively (Figure 4). There was a significant difference in percentage of brain wet matter between the control group and the groups euthanized 6 and 24 hours after injection of glyceryl trinitrate. However, there was no significant difference between the 2 groups injected with glyceryl trinitrate. Results confirmed that there was brain edema 6 and 24 hours after injection of glyceryl trinitrate.
Box-and-whisker plots of brain edema as determined by use of the wet-dry weight method for control carp and carp injected IP with glyceryl trinitrate (1 mg/kg) and euthanized 6 and 24 hours after injection (n = 15 fish/group). Each box represents the 25th and 75th percentiles, the horizontal line in each box represents the median, and the whiskers represent the minimum and maximum. *Value differs significantly (P ≤ 0.05) from the value for the control group.
Citation: American Journal of Veterinary Research 76, 7; 10.2460/ajvr.76.7.615
Brain edema and vasodilation determined by use of H&E-stained brain preparations
Histologic examination of H&E-stained sections of the brains of carp euthanized 6 and 24 hours after injection of glyceryl trinitrate revealed signs of brain edema and vasodilation in all fish. The most noticeable findings were sponge-like changes visible as perivascular and pericellular vacuolization (Figure 5). Furthermore, other generally accepted morphological criteria of cerebral edema (pallor of myelin, loose or sieve-like appearance of myelinated areas, and rarefaction of subpial spaces) were assessed. Dilated blood vessels filled with blood and thin, elongated, but regular endothelial cells indicated that vasodilation was also evident in the glyceryl trinitrate-treated fish. No histopathologic changes related to brain edema or vasodilation were found in carp of the control group.
Photomicrograph of sections of the brain of a representative carp obtained 6 hours after IP injection of glyceryl trinitrate. Visible signs of edema are evident, including sponge-like changes in pericellular (circles) and perivascular (stars) tissues. Notice the dilated blood vessels filled with blood and thin, elongated, but regular endothelial cells (arrows). H&E stain; bar = 100 μm.
Citation: American Journal of Veterinary Research 76, 7; 10.2460/ajvr.76.7.615
Discussion
In the present study, effects of glyceryl trinitrate on BBB permeability of carp were evaluated. In all mammals that have been examined, NO donors cause an increase in BBB permeability.7,24,25 Nitric oxide synthase is found in the CNS of all vertebrates, but the role and function of NO has not been precisely determined, especially for ectothermic animals.26 The manner in which NO affects permeability of the BBB in lower vertebrates is not known. In a previous study18 conducted by our research group, carp were evaluated 1, 3, 6, and 8 hours after administration of glyceryl trinitrate treatment; there was increased permeability of the BBB in the brain tissue for the group evaluated 6 hours after injection. In the present study, we determined the nitrate and nitrite concentrations in carp serum up to 24 hours after injection of glyceryl trinitrate. Analysis of the results revealed a biphasic pattern of changes with a significant increase in concentrations 0.25 and 1 hour after injection of glyceryl trinitrate, which was followed by a decrease in concentrations to values similar to those for the control group. The highest concentrations were detected 6 and 8 hours after injection of glyceryl trinitrate, whereas concentrations returned to values similar to those of the control group by 12 hours after injection. The increase in nitrate and nitrite concentrations in the groups evaluated 6 and 8 hours after injection indicated possible endogenous NO synthesis induced by the NO donor glyceryl trinitrate. The serum nitrite concentration was significantly lower than the serum nitrate concentration. Even though the concentrations of nitrate and nitrite should be almost identical because nitrate is converted to nitrite, there is almost always a difference in concentrations in vivo because of incomplete reduction to nitrite.27
Trace quantities of NO are produced by neurons, endothelial cells, platelets, and neutrophils in response to homeostatic stimuli.28,29 This NO is scavenged rapidly (half-life, 4 seconds) and acts in a paracrine manner to transmit cellular signals. Nitric oxide is also produced by other cells (macrophages, fibroblasts, and hepatocytes) in micromolar concentrations in response to inflammatory or mitogenic stimuli. In those situations, the biological role for NO is defense against non-self-pathogens through oxidative toxicosis. These high NO concentrations lead to the formation of peroxynitrite. Thus, the amount of NO produced by various biological systems can differ by several orders of magnitude, and the subsequent chemical reactivity of NO is diverse. Nitric oxide undergoes a series of reactions with molecules in biological fluids, but the final products of NO in vivo are nitrite and nitrate. The relative proportion of nitrate and nitrite is variable and cannot be predicted with certainty.30 Thus, the best index of total NO production is the sum of both nitrate and nitrite concentrations.31 Results of the present study confirmed that the ratio of nitrate to nitrite is quite variable, and the total nitrate and nitrite concentration appeared to be the best indicator of NO formation.
Glyceryl trinitrate is an organic nitrate with a short plasma half-life (1 to 4 minutes), but on the basis of results of tissue concentrations of glyceryl trinitrate and cyclic guanosine monophosphate after administration to rats in vivo, it has been proven that its half-life can be as much as 2 hours.32,33 Glyceryl trinitrate metabolizes to NO within a cell through the actions of glutathione S-transferase, cytochrome P450, and the thiol reaction.34 On the basis of an increase in the total serum nitrate and nitrite concentration for fish evaluated 6 and 8 hours after injection of the NO donor, we believe that there was endogenous synthesis of NO initiated by glyceryl trinitrate, which was suppressed by 12 and 24 hours after injection of glyceryl trinitrate. Thus, both the previous study18 conducted by our research group and the study reported here indicate that administration of an NO donor can trigger the induction of endogenous NO synthesis by NO synthases.35–37 The observed biphasic pattern for the measured NO concentration after injection of glyceryl trinitrate is in accordance with results of another study.38
Evans blue dye is an azo dye with a great affinity for serum albumin and typically is used for intravital assessment of BBB permeability. Results for the group of fish euthanized 6 hours after injection of glyceryl trinitrate provided evidence of leakage of Evans blue dye, which indicated increased permeability of the BBB. However, analysis of results for the groups euthanized 12 and 24 hours after injection revealed no significant differences in staining with Evans blue dye, compared with results for the control group, which suggested that the BBB was again impermeable and functioning normally.
Experiments in the present study to evaluate brain edema revealed signs of edema in both glyceryl trinitrate-treated groups evaluated 6 and 24 hours after injection. Despite the fact the BBB was impermeable, as determined by impermeability to albumin molecules complexed with Evans blue dye, excessive fluid remained trapped in the interstitial part of the brain up to 24 hours later.
Numerous studies39–41 have been performed on mammals to determine associations of increased BBB permeability and the appearance of cerebral edema with increased NO synthesis. In the study reported here, a time connection existed between the total serum nitrate and nitrite concentration in carp and BBB permeability (degree of intensity of staining with Evans blue dye) as well as between the total serum nitrate and nitrite concentration and brain edema formation. However, the data did not yield precise information on whether the role of NO in brain edema formation was indirect (ie, whether the edema was a consequence of BBB permeability under the influence of NO) or whether NO directly participated in brain edema formation independently from its effect on BBB permeability. Investigators of other studies42,j found that administration of an inhibitor of inducible NO synthase reduces neurologic deficits but not cerebral edema, which appears to be a consequence of traumatic brain damage. Analysis of the results of this and other studies40–42 does not support a direct role of NO in brain edema formation.
The effects of an NO donor on cerebral vasodilation were evaluated in the present study. Results reported here, as well as results of numerous other studies,16,43–45 confirm the role of NO in the increase of cerebral blood flow for higher and lower vertebrates.
Evidence suggests that barrier layers at the key interfaces between blood and neural tissue surrounding the central synapses were one of the chief evolutionary pressures leading to the development of mechanisms for maintaining homeostasis of the neural microenvironment.46 The barrier function is not fixed and can be modulated and regulated with numerous signaling processes (both physiologic and pathological). Modulating agents can originate from blood or from the cells associated with blood vessels. Nitric oxide is an important molecule in blood circulation of the brain, but its function differs depending on the evolutionary degree of the species.47 The number of comparative physiologic studies regarding the precise role and function of NO in certain types of lower vertebrates is insufficient. In fish, the greatest attention has been given to evaluation of cerebral blood flow regulation by NO.16 In the present study, we examined the influence of NO on BBB permeability in carp, which thus contributed to the body of knowledge on NO and its effects in lower vertebrates.
Extravascular outflow of protein serum or vasogenic brain edema is inevitable when there is increased permeability of the BBB.48,49 The amount of time during which there is increased permeability of the BBB is an important factor for the severity of brain edema.50 Increased permeability of the BBB leads to the leakage of plasma proteins into the interstitial space, leukocyte migration into the CNS, and development of an inflammatory reaction.51 Brain edema formation is followed by an increase in blood pressure, which leads to a decrease in cerebral blood flow, a subsequent decrease of the energy supply, and further tissue damage.52
Analysis of results for the study reported here suggested that administration of an NO donor can cause a reversible and time-predictable increase in permeability of the BBB in common carp. Consequently, brain edema and vasodilation occur. The time course of the alterations in these lower vertebrates is similar to changes reported for several mammalian species.
Acknowledgments
This manuscript represents a portion of a thesis submitted by Dr. Kovacic to the University of Zagreb School of Medicine as partial fulfilment of the requirements for a Doctor of Science degree.
Supported by a research grant from the Croatian Ministry of Science and Technology (project No. 006-0061117-1242).
The authors have no competing interest, no financial relationships during the previous 3 years with organizations that might have an interest in the submitted manuscript, and no other relationships or activities that could have influenced the submitted manuscript.
The authors thank Gordana Husinec for assistance with fish handling.
ABBREVIATIONS
| BBB | Blood-brain barrier |
| NO | Nitric oxide |
Footnotes
Lach-Ner sro, Neratovice, Czech Republic.
Aller Aqua, Christiansfeld, Denmark.
SevenGo Duo Pro probe, Mettler Toledo GmBH, Greifensee, Switzerland.
HI700 ammonia checker, Hanna Instruments, Woonsocket, RI.
Sigma Aldrich Chemie, Munich, Germany.
Trinitrosan, Merck Pharma, Darmstadt, Germany.
Millipore Co, Billerica, Mass.
Cayman Chemical Co, Ann Arbor, Mich.
SPSS, version 11.5, IBM, Chicago, Ill.
Louin G, Marchand-Verrecchia C, Palmier B, et al. Effect of three inducible NOS inhibitors on cerebral edema formation and neurological deficit in a rat model of traumatic brain injury (abstr).J Cereb Blood Flow Metab 2005;25:S49.
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