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A brain-slicing apparatus for tissue respiration studies
ANALYTICAL
BIOCHEMISTRY
A Brain-Slicing
13, 468-462
Apparatus
SOMASUNDARAM AND From
the Departments The College Children’s
(1965)
for
Tissue
Respiration
ADDANKI, JUAN PHILLIP D. REARICK
of
Studies1
F. SOTOS,
of Pediatrics and Physiological Chemistry, Medicine, Ohio State University, and Hospital Research Foundation, Columbus, Ohio Received
July
12, 1965
The problem of preparation of brain slices with a minimum damage to the tissue for respiration studies has never been adequately solved. Brain tissue is softer than other tissues of the body and is thus subjected to more maceration and damage when employing the free-hand method of cutting slices. The problem is still worse in the small animal such as the mouse or the rat where the organ is not only friable but is also small. Three different brain-slicing apparatuses were described in the past (l-3). However, each one described has one or more limitations, such as the complexity of design (l), difficulty in handling and cutting the slices viewing from the top of a moist cold box (2), the expensiveness of the apparatus (3) and, more so, that some of them are not available commercially. In connection with an investigation requiring brain tissue slices for respiration, the following slicing apparatus was devised and is being used successfully, In general, the procedure consists of holding the brain tissue in a well formed by the aligned holes of the templates in a cold room at 4°C and pouring the liquid agar (1%) at 37°C around the tissue in the well. When the entire apparatus with the brain tissue and agar is left for 5 min in the cold room, the agar solidifies and encompasses the tissue in such a way that the removal of templates one at a time furnishes a hard protruding mass to be cut with a knife. DESCRIPTION
All the components are made with stainless steel.? These (Fig. 1) consist of a base plate, a resting template, active templates, two springs, ‘This investigation was supported by Public Health NB-05114 and FR-78 from the Institute of Neurological Clinical Research Branch. ‘Made by Partsco, Inc., Columbus, Ohio, according 458
Service Research Grant Diseases and Blindness to our
specifications.
No. and
BRAIN-SLICING
APPARATUS
459
FIG :. 1. Various components of the slicing apparatus. Base plate vcith blr mk t;emplate (bof ;tom most), active templates (middle) with lateral tabs at thl *ee diffe rent locati oils. Springs and thumbscrew (top). Stradie and Riggs microt ,oml e k:nife (on right) (se6 3 text for exact measurements).
and two thumbscrews. The base plate (177 X 101 X 12 mm) has two vertical circular posts (o.d. = 7 mm) 76 mm long, fixed at 101 mm apart along the center line and 38 mm from either end of the base plate. The resting template (127 X 50 X 0.5 mm) is located next to the base plate. All the active templates (127 X 50 X 0.5 mm) have four holes, two small holes (o.d. = 7 mm) along the center line of the templates at 101 mm apart and 12 mm from either end. These two small holes, in the normal operation, slip onto the two vertical posts of the base plate and align the two larger holes (o.d. = 19 and 25 mm) of the other active templates forming two wells (o.d. = 19 and 25 mm). Also, the active templates have two lateral tabs either diagonally or centrally located to facilitate
460
ADDANKI,
SOTOS,
AND
REARICK
easy removal. The springs are designed to press and hold tight the templates in place with the thumbscrews. A Stradie and Riggs (4) microtome knife was used to cut the slices.
FIQ. 2. View of assembled slicing apparatus with brain tissue and agar in situ while making the slices. METHODS
Figure 2 illustrates the entire apparatus with the brain tissue and agar in situ when assembled for the preparation of brain slices. All the procedures described were performed in a temperature-controlled room at 4%. The actual procedure begins by removing and transferring the brain tissue to one of the wells and holding it in a desired position with a forceps while the liquid agar (1%) at 37°C (1) is poured around the tissue. By leaving the entire apparatus with the tissue and agar in situ for 5 min, the agar solidifies, holding the brain tissue firmly within the well. By removing one template, the protruding part of the agar containing the brain tissue can be cut with a knife. By repeating this procedure, a battery of slices can be prepared, each slice having the same thickness as that of the templates (0.5 mm) removed. Two wells are provided for the use of either two lobes of one brain or two different brains depending upon the type of the experiment. The entire slicing procedure takes no more than 15 min for two different rat brains. Excess agar is removed from each slice by teasing with a pointed needle and the slice is then weighed and transferred into a Warburg flask containing the medium. The brain slices prepared as described were incubated in a Warburg respirometeP with 3 ml of Krebs-Ringer phosphate buffer at pH 7.4 (5) a Model
WB3
Gilson Medical
Electronics,
Middleton,
Wisconsin.
BRAIN-SLICI~W
461
APPARATUS
containing 37.5 pmoles of glucose. In the center well of the reaction flask 0.2 ml of 10% KOH was placed. All the flasks were gassed with 100% oxygen for 10 min and their respiration was measured for the next 9 hr. RESULTS
AND
DISCUSSION
In order to study the variation in t’hickness of the brain slices, the thickness of 19 brain slices was calculated from the weight, density, and area of the slice. The mean thickness was found to be 0.60 mm, with a standard error of the mean of 0.02 mm. The mean thickness obtained was within the permissible range as calculated by Field (6). Histological preparations of brain slices cut at right angle to the original section to demonstrate the edge of the sliced tissue were examined. A study of these sections indicated that tearing and distortion of the tissue slices were minimal. The integrity of the cell structures along t,he cut surface appears to be preserved.
RESPIRATIONS
OF RAT
Time, hr 1 2 3 4 5 6 7 8 9 0 Average of 10 determinations. * S.D. = standard deviation
TABLE 1 BRAIN SLICES 02, &Lmoles 8.14 15.77 23.19 29.79 36.19 42.03 47.13 51.90 56.16
(100
MG
WET
WEIGHT) F5.D.b 0.49 1.00 1.48 1.95 2.22 2.65 3.01 3.36 4.32
of the observations.
Table 1 indicates the pmoles of Oz taken up per 100 mg wet tissue at different time intervals. The table clearly demonstrates that the brain slices respired linearly for at least 4 hr, after which they respired at a slightly lowered rate. The values obtained for the first 4 hr are similar to those of Crismon et al. (2), who reported 8.9 pmoles of 0, taken up per 100 mg wet brain tissue per hour when their data was calculated to liken our measurements. Also, this apparatus has been used for making slices of liver tissue with the same precision in the technique. Instead of stainless-steel templates, templates made from Plexiglas with identical measurements, were also used successfully in our initial experiments.
462
ADDANRI,
SOTOS,
AND
REARICK
SUMMARY
An apparatus is described which is simple in design, easy to operate, and needs no training or experience for preparing brain slices of uniform thickness (0.6 mm) for tissue respiration studies. Histological examination of the rat brain slices made indicated that the integrity of the cell structures appear to be preserved. These brain slices respired linearly for the first 4 hr of incubation at a rate similar to that reported in the literature. ACKNOWLEDGMENTS The authors wish to thank Dr. Charles B. Reiner, Pathologist, Children’s Hospital, for his valuable assistance in the preparation and microscopic evaluation of the brain slices, and Mr. Rudolph Levandofsky for drawing the blueprint of the slicing apparatus. REFERENCES 1. GRACA, G. J., AND MAKAROFF, N. W., Science 115,374 (1952). CRISMON, J. M., AND FIELD, J., II, Am. J. Physiol. 130, 231 (1940). MCILWAIN, H., AND BCJDDLE, H. L., Biochem. J. 53,412 (1953). STRALJIE, W. C., AND RIGGS, B. C., J. Biol. Chem. 154, 687 (1944). UMBRE~T, W. W., BURRIS, R. H., AND STAUFFER, J. H., in ‘LManometric Techniques,” p. 132. Burgess Publishing Company, Minnesota, 1964. 6. FIELD, J., II, in “Methods of Medical Research” (V. R. Potter, ed.), Vol. 1, p. 289. Year Book Publishers, Chicago, 1948. 2. 3. 4. 5.
BIOCHEMISTRY
A Brain-Slicing
13, 468-462
Apparatus
SOMASUNDARAM AND From
the Departments The College Children’s
(1965)
for
Tissue
Respiration
ADDANKI, JUAN PHILLIP D. REARICK
of
Studies1
F. SOTOS,
of Pediatrics and Physiological Chemistry, Medicine, Ohio State University, and Hospital Research Foundation, Columbus, Ohio Received
July
12, 1965
The problem of preparation of brain slices with a minimum damage to the tissue for respiration studies has never been adequately solved. Brain tissue is softer than other tissues of the body and is thus subjected to more maceration and damage when employing the free-hand method of cutting slices. The problem is still worse in the small animal such as the mouse or the rat where the organ is not only friable but is also small. Three different brain-slicing apparatuses were described in the past (l-3). However, each one described has one or more limitations, such as the complexity of design (l), difficulty in handling and cutting the slices viewing from the top of a moist cold box (2), the expensiveness of the apparatus (3) and, more so, that some of them are not available commercially. In connection with an investigation requiring brain tissue slices for respiration, the following slicing apparatus was devised and is being used successfully, In general, the procedure consists of holding the brain tissue in a well formed by the aligned holes of the templates in a cold room at 4°C and pouring the liquid agar (1%) at 37°C around the tissue in the well. When the entire apparatus with the brain tissue and agar is left for 5 min in the cold room, the agar solidifies and encompasses the tissue in such a way that the removal of templates one at a time furnishes a hard protruding mass to be cut with a knife. DESCRIPTION
All the components are made with stainless steel.? These (Fig. 1) consist of a base plate, a resting template, active templates, two springs, ‘This investigation was supported by Public Health NB-05114 and FR-78 from the Institute of Neurological Clinical Research Branch. ‘Made by Partsco, Inc., Columbus, Ohio, according 458
Service Research Grant Diseases and Blindness to our
specifications.
No. and
BRAIN-SLICING
APPARATUS
459
FIG :. 1. Various components of the slicing apparatus. Base plate vcith blr mk t;emplate (bof ;tom most), active templates (middle) with lateral tabs at thl *ee diffe rent locati oils. Springs and thumbscrew (top). Stradie and Riggs microt ,oml e k:nife (on right) (se6 3 text for exact measurements).
and two thumbscrews. The base plate (177 X 101 X 12 mm) has two vertical circular posts (o.d. = 7 mm) 76 mm long, fixed at 101 mm apart along the center line and 38 mm from either end of the base plate. The resting template (127 X 50 X 0.5 mm) is located next to the base plate. All the active templates (127 X 50 X 0.5 mm) have four holes, two small holes (o.d. = 7 mm) along the center line of the templates at 101 mm apart and 12 mm from either end. These two small holes, in the normal operation, slip onto the two vertical posts of the base plate and align the two larger holes (o.d. = 19 and 25 mm) of the other active templates forming two wells (o.d. = 19 and 25 mm). Also, the active templates have two lateral tabs either diagonally or centrally located to facilitate
460
ADDANKI,
SOTOS,
AND
REARICK
easy removal. The springs are designed to press and hold tight the templates in place with the thumbscrews. A Stradie and Riggs (4) microtome knife was used to cut the slices.
FIQ. 2. View of assembled slicing apparatus with brain tissue and agar in situ while making the slices. METHODS
Figure 2 illustrates the entire apparatus with the brain tissue and agar in situ when assembled for the preparation of brain slices. All the procedures described were performed in a temperature-controlled room at 4%. The actual procedure begins by removing and transferring the brain tissue to one of the wells and holding it in a desired position with a forceps while the liquid agar (1%) at 37°C (1) is poured around the tissue. By leaving the entire apparatus with the tissue and agar in situ for 5 min, the agar solidifies, holding the brain tissue firmly within the well. By removing one template, the protruding part of the agar containing the brain tissue can be cut with a knife. By repeating this procedure, a battery of slices can be prepared, each slice having the same thickness as that of the templates (0.5 mm) removed. Two wells are provided for the use of either two lobes of one brain or two different brains depending upon the type of the experiment. The entire slicing procedure takes no more than 15 min for two different rat brains. Excess agar is removed from each slice by teasing with a pointed needle and the slice is then weighed and transferred into a Warburg flask containing the medium. The brain slices prepared as described were incubated in a Warburg respirometeP with 3 ml of Krebs-Ringer phosphate buffer at pH 7.4 (5) a Model
WB3
Gilson Medical
Electronics,
Middleton,
Wisconsin.
BRAIN-SLICI~W
461
APPARATUS
containing 37.5 pmoles of glucose. In the center well of the reaction flask 0.2 ml of 10% KOH was placed. All the flasks were gassed with 100% oxygen for 10 min and their respiration was measured for the next 9 hr. RESULTS
AND
DISCUSSION
In order to study the variation in t’hickness of the brain slices, the thickness of 19 brain slices was calculated from the weight, density, and area of the slice. The mean thickness was found to be 0.60 mm, with a standard error of the mean of 0.02 mm. The mean thickness obtained was within the permissible range as calculated by Field (6). Histological preparations of brain slices cut at right angle to the original section to demonstrate the edge of the sliced tissue were examined. A study of these sections indicated that tearing and distortion of the tissue slices were minimal. The integrity of the cell structures along t,he cut surface appears to be preserved.
RESPIRATIONS
OF RAT
Time, hr 1 2 3 4 5 6 7 8 9 0 Average of 10 determinations. * S.D. = standard deviation
TABLE 1 BRAIN SLICES 02, &Lmoles 8.14 15.77 23.19 29.79 36.19 42.03 47.13 51.90 56.16
(100
MG
WET
WEIGHT) F5.D.b 0.49 1.00 1.48 1.95 2.22 2.65 3.01 3.36 4.32
of the observations.
Table 1 indicates the pmoles of Oz taken up per 100 mg wet tissue at different time intervals. The table clearly demonstrates that the brain slices respired linearly for at least 4 hr, after which they respired at a slightly lowered rate. The values obtained for the first 4 hr are similar to those of Crismon et al. (2), who reported 8.9 pmoles of 0, taken up per 100 mg wet brain tissue per hour when their data was calculated to liken our measurements. Also, this apparatus has been used for making slices of liver tissue with the same precision in the technique. Instead of stainless-steel templates, templates made from Plexiglas with identical measurements, were also used successfully in our initial experiments.
462
ADDANRI,
SOTOS,
AND
REARICK
SUMMARY
An apparatus is described which is simple in design, easy to operate, and needs no training or experience for preparing brain slices of uniform thickness (0.6 mm) for tissue respiration studies. Histological examination of the rat brain slices made indicated that the integrity of the cell structures appear to be preserved. These brain slices respired linearly for the first 4 hr of incubation at a rate similar to that reported in the literature. ACKNOWLEDGMENTS The authors wish to thank Dr. Charles B. Reiner, Pathologist, Children’s Hospital, for his valuable assistance in the preparation and microscopic evaluation of the brain slices, and Mr. Rudolph Levandofsky for drawing the blueprint of the slicing apparatus. REFERENCES 1. GRACA, G. J., AND MAKAROFF, N. W., Science 115,374 (1952). CRISMON, J. M., AND FIELD, J., II, Am. J. Physiol. 130, 231 (1940). MCILWAIN, H., AND BCJDDLE, H. L., Biochem. J. 53,412 (1953). STRALJIE, W. C., AND RIGGS, B. C., J. Biol. Chem. 154, 687 (1944). UMBRE~T, W. W., BURRIS, R. H., AND STAUFFER, J. H., in ‘LManometric Techniques,” p. 132. Burgess Publishing Company, Minnesota, 1964. 6. FIELD, J., II, in “Methods of Medical Research” (V. R. Potter, ed.), Vol. 1, p. 289. Year Book Publishers, Chicago, 1948. 2. 3. 4. 5.