Since the first time-sharing light-guide fluorometer was built at the end of 1972, fiber-optics have been used in various types of fluorometers [4, 5]. The “direct-current” (DC) fluorometer–reflectometer containing a Y-shaped light guide has been of value in most of the studies in which NADH fluorescence was measured. Mayevsky and Bar-Sagie [6] described the use of the two-channel DC fluorometer–reflectometer with dual Y-shaped light guides in the study of brain energy metabolism (Fig. 6.2b). The localization of the probes on the brain are shown in Fig. 6.2a as presented by Zarchin and Mayevsky in 1981 [7]. In addition to the two NADH probes, four ECoG electrodes provide the electrical activities of the two hemispheres. Also, a special push-pull cannula was located on each hemisphere to elicit cortical spreading depression (SD) by high level of KCl if needed. Typical responses to SD, elicited at the same time, are shown in Fig. 6.3 [6]. The lower part was measured after topical application of glucose to the left hemisphere while the upper part was monitored after exposure to 2-deoxyglucose. Oxidation of NADH during SD was attenuated after treatment with 2-deoxyglucose.

The responses of the two hemispheres to decapitation (complete ischemia) (Fig. 6.4) are discussed in detail by Zarchin and Mayevsky in 1981 [7]. The responses of the two hemispheres were very similar in the two monitored areas.

The use of the two-channel fluorometer was very significant when changes in blood supply were studied in the gerbil brain. Figure 6.5a illustrates the anatomy of the arteries providing blood to the brain of the gerbil, including the circle of Willis (COW). Details have been discussed in a few papers [8, 9]. Figure 6.5b, c shows the complete COW after perfusion and fixation of the blood vessels. In Fig. 6.5b, the two optic nerves were removed to better present the blood vessels.

The optical probes were located above the two hemispheres of the gerbil exposed to anoxia (Fig. 6.6a). Under anoxia (100 % N₂), NADH was elevated similarly in the two hemispheres as seen in the CF signals. The effects of unilateral carotid artery occlusion are presented in Fig. 6.6b, c. When the left carotid artery was occluded (b), NADH was elevated only in the occluded side. The same response was recorded when the right carotid artery was occluded (c). In this specific gerbil, the circle of Willis was disconected in the anterior cerebral arteries area. This figure indicates the value of NADH monitoring to study anatomical issues.

Figure 6.7a shows the response to unilateral, bilateral occlusion as well as the addition of N₂ to the occluded brain [9]. Under right occlusion the right-hemisphere NADH shows a transient increase followed by slow recovery caused by the compensation of blood flow via the anterior vertebral artery. The left hemisphere shows a very small transient increase. The addition of N₂ did not affect the redox state because of the lack in posterior communication. The same results were obtained when the left carotid artery was occluded initially followed by right artery occlusion (Fig. 6.7b).

Monitoring of NADH in the blood-perfused beating heart at a single point was done in combination of measuring local myocardial perfusion and local contractile activity in a circumscribed area of the left ventricular surface in the open chest dog preparation [10–12]. Measurement of tissue blood flow (by a thermistor technique) gives an indication of the oxygen delivery to that area of the myocardium. Under in vivo conditions, NADH fluorescence is inversely proportional to the oxygen concentration in the mitochondria because NAD is the first hydrogen acceptor in the respiratory chain. The isometric tension developed by contraction of local muscle cells can be considered to represent the O₂ demand of those cells. Thus, the simultaneous and continuous measurement of these three parameters makes it possible now to evaluate myocardial oxygen balance and to characterize the effect of various physiological conditions upon it. The technological aspects of the monitoring system were described in Sect. 4.​9.​1.​3.

In more advanced studies, three parameters were measured at two different sites on the left ventricular wall: isometric force of contraction, local myocardial blood supply, and intramitochondrial NADH fluorescence. One set of transducers was placed on the anterior surface toward the apex, below the main bifurcation of the left anterior descending coronary artery. The other set was sutured close to the base of the left ventricle and left of the origin of the anterior descending coronary artery, directly below the origin of the circumflex coronary artery. The exact placement is shown in Fig. 6.8.

Figure 6.9a, b presents polygraph tracings [13] comparing the effect of changes in heart rate upon the parameters measured immediately preceding coronary ligation (Fig. 6.9a) with those following it (Fig. 6.9b). The upper five tracings are taken from the lower part of the left ventricle (directly below the ligation site) and the lower five tracings were recorded from the base of the left ventricle (nonischemic area). The left panel shows that preceding ligation, an increase in heart rate from 120/min to 180/min caused a 40–60 % elevation in NADH fluorescence followed by a 130–140 % elevation of local coronary blood supply to both areas of the heart. Further elevation in stimulus frequency to 240/min produced similar effects, although coronary vasodilation was apparently less marked at the lower area of the heart than at its base. Isometric contractile force did not decrease significantly at the higher ventricular rates.

Following coronary ligation, there was an immediate decrease in T flow to the ischemic area by 60 %, and also a 160 % elevation in NADH fluorescence. Approximately 10 min later, changes in heart rate were produced (Fig. 6.9a). Figure 6.9b shows that following ligation the ischemic area was no longer capable of coronary vasodilation as heart rate was increased from 120/min to 180/min; a decrease in local blood supply was observed. A small increase in NADH fluorescence can be detected, and contractile force was not diminished. Further increase in ventricular rate to 240/min resulted in a marked decrease in contractile force with little further change in NADH fluorescence and T flow. In contrast, the ‘nonischemic’ area preserved the capacity for coronary vasodilation with increasing heart rate, and NADH levels increased with heart rate in a manner similar to that observed before ligation. Nevertheless, several differences can be seen in the nonischemic area following ligation. Coronary vasodilation produced by increased heart rate was less pronounced following ligation, and the increase in NADH fluorescence following abrupt elevation of heart rate was greater. Contractile force was diminished at 240/min.

Use of the four-channel DC fluorometer–reflectometer to monitor four different locations on the same brain was the next step in our technological development. In this fluorometer we used one light source to illuminate simultaneously the four monitored areas in the same brain. This arrangement avoids the possible differences in the excitation light intensity delivered to the four points and therefore decreases the variation in the result measured from the four sites when exposed to the same treatment. Figure 6.10 shows the schematics of the monitoring system [9]. The emitted light from the four monitoring sites is collected separately and treated in four identical detection and amplification units. The four light guide arms could be placed in the same hemisphere or in both hemispheres of the brain.

Figure 6.11 shows a typical response of the gerbil brain to anoxia (a) and induced cortical spreading depression (b) [2]. These responses were typical and were found in all normoxic gerbil brains tested. The animal was breathing air spontaneously, and when it was exposed to 100 % nitrogen, a typical two-step decrease in reflectance was recorded together with a large increase in corrected NADH fluorescence, which reached a plateau shortly thereafter. When air breathing was restored, rapid oxidation of NADH was recorded in all four sites. In this gerbil the anoxia induced also a secondary response characterized by an oxidation cycle of NADH that appeared first in site 1, then propagated to sites 2 and 3, and finally reached site 4. This phenomenon is a spreading depression wave initiated during the anoxia by the intrinsic elevated extracellular K⁺ ions as was found when we monitored only one site [14]. The same response to spreading depression was monitored after initiation of a wave by application of KCl solution via a special cannula placed anteriorly to the monitored sites (Fig. 6.11b). When the wave reached site 1 (the one closest to the KCl application site), a typical biphasic change in the reflected light was recorded in the four monitored sites. The NADH-corrected fluorescence showed an oxidation cycle lasting between 1 and 2 min, depending on the measuring site in relationship to the propagation front of the spreading depression wave. The reflectance response had a short increase phase, followed by a very long decrease below baseline, and then a recovery phase.

To study the propagation speed of the CSD wave, a second type of light guide holder was constructed; the results were presented in 1983 [15]. The light guides were organized along the KCl application site. The distance between site 1 and 4 was 5 mm and the difference in the responses was about 130 s, so the speed in this gerbil was 2.3 mm/min.

The four-channel fluorometer was used in studies where the anatomy of blood vessels in the gerbil brain was compared to the NADH responses to ischemia. In these studies [3, 9], we used the system presented in Fig. 6.10 and the four monitoring sites were as presented in the lower left side of the scheme.

Figure 6.12 shows a typical Meriones unguiculatus (Mongolian gerbil) response to various perturbations while simultaneously monitoring the NADH from four sites (Fig. 6.10). The anatomical pattern of this gerbil determined that it was typical of an A _II vasculature, namely, that the connection between the two anterior cerebral arteries (ACAs) was incomplete (Fig. 6.5b). Figure 6.5a demonstrates that all four monitoring sites responded similarly to anoxia [9] as evaluated by the corrected NADH fluorescence (CF₁–CF₄) as well as by the reflectance (R₁–R₄). When the right carotid artery was occluded (R_OC), sites 1 and 2 measuring from the right hemisphere exhibited a clear increase in NADH (CF1 + CF2). Site 3, measuring from the midline area of the left hemisphere, also exhibited an increase in NADH, whereas site 4 was unchanged. The same pattern of responses was found when the left carotid artery was occluded initially (Fig. 6.12c). As expected, all four sites showed an increase in NADH during the bilateral occlusion (L_OC in part b or R_OC in part c). Exposure of the bilateral occluded gerbil to N₂ did not result in a further significant increase in NADH. Some sites showed a response to spreading depression, which developed during the ischemic-anoxic event during the recovery period. These results suggest that site 3 was supplied via the same arteries as sites 1 and 2, although it is located on the contralateral hemisphere.

When the same type of monitoring was performed using a rat, no difference was found in the response to unilateral occlusion between the four monitored sites.