0361-9230/91 $3.00 + .OO
Brain Research Bulletin, Vol. 27, pp. 283-286. Q Pergamon Press plc, 1991. Printed in the U.S.A.
BRIEF COMMUNICATION
Changes in Physiological Parameters of Rat Cerebrospinal Fluid During Chronic Sampling: Evaluation of Two Sampling Methods IRENA WESTERGREN Department
of Neurology,
AND BARBRO
B. JOHANSSON
University Hospital, S-221 85 Lund, Sweden
Received
23 October
1990
WESTERGREN, I. AND B. B. JOHANSSON. Changes in physiological parameters of rat cerebrospinal fluid during chronic sampling: Evaluation of two sampling methods. BRAIN RES BULL 27(2) 283-286, 1991.-Although rat cerebrospinal fluid (CSF) is increasingly being used in pharmacological and biochemical research, methodological studies on basic physiological data are lacking. We have determined the albumin content and number of erythrocytes and leukocytes in CSF obtained by two different methods of sampling from cistema magna-repeated sampling from an implanted cannula and by repeated punctures. In the initial samples the albumin content was 0.08*0.03 pg/pl. Chronic cannulation of the cistema magna resulted in a meningeal reaction with increased cell and albumin content: a reaction that could be reduced but not prevented by using a sterile cannula. The number of leukocytes but not erythrocyteswas highly correlated to the albumin content. Repeated sampling in the absence of a permanent cannula did not significantly increase albumin content but carried a higher risk for erythrocyte contamination. CSF
Rat
Albumin
Erythrocytes
Leukocytes
(Hamilton Co. Inc., Whittier, CA 710). The tip of the needle perfectly fit the upper end of the cannula without leaving any dead space, thus the amount of CSF withdrawn could be controlled.
IN spite of the common use of rat cerebrospinal fluid in pharmacological studies, no data is available on the physiological levels of albumin and cell content, nor the possible effect of chronic cannulation on these parameters. Sampling of CSF from cistema magna in rat is usually not performed under sterile conditions. Following earlier described techniques (l-3, 6, 8-l l), we have in preliminary studies observed a considerable meningeal reaction. As a base for further studies on blood-CSF and brainCSF transfer under normal and pathological conditions, we have therefore determined the physiological baseline value for albumin and cells and to what extent these parameters can be altered during chronic cannulation or repeated samplings.
Implantation
of the Cannula and CSF Sampling
Male Sprague-Dawley rats (250-300 g body weight) were anesthetized with methohexital (Brietal) 60 mg/kg IP. The head was fixed in horizontal position in a stereotaxic apparatus, the incision area shaved and a l-cm midline incision was made in the skin to expose the dorsal and posterior surfaces of the skull. A hole was drilled with a small dental drill at the sag&al midline through the suture between the interparietal and occipital bone at 60” angle to the interparietal bone. So far the procedure was identical for all rats. The rats were divided into 2 groups.
METHOD
The tips of Venisystems Butterfly-21G steel needles (Abbot Scandinavia AB, 16421 Kista) were cut to a length of 7 mm from the point of insertion. The obturator from B-D spinal needles 90-7 (Becton, Dickinson and Company, Rutherford, NJ 07070) was cut to a length to make it just visible on the tip when inserted into the upper end of the needle. The lateral wings of the infusion set were reduced. The needle (Fig. 1A) was used as a chronically implanted cammla as well as for repeated cannulation. The cannula was either cleaned with physiological solution (nonsterile cannula) or sterilized by retaining it in 95% ethanol for 24 hours and flushed with sterile physiological solution before use. CSF was drawn with a lOO+l Hamilton needle
Rats With Indwelling Cannulae
Two additional holes were drilled in the interparietal bone for placement of screws. The obturator was placed into the cannula which was then inserted to its entire depth (i.e., 7 mm from the outer surface of the skull) into the prepared opening at approximately 60” angle to the interparietal bone by free hand. The position of the cannula in cistema magna (Fig. 1C) was controlled by removal of the obturator, insertion of the tip of the Hamilton needle and withdrawal of CSF. The cannula was then anchored
283
284
WESTERGREN
AND JOHANSSON
TABLE 1 ALBUMIN CONTENT IN RAT CISTERNAL FLUID @&I) DURING REPEATED SAMPLING WITH OR WITHOUT PERMANENT CANNULATION Experimental Group
Sample 1
2
3
J
-__
FIG. 1. The cannula tema magna (C).
(A), metal stop (B) and cannula’s
position in cis-
to the skull and to the screws with acrylic dental cement and closed with a metal stop (made by the local research technical department, Fig. 1B) placed in the upper end of the needle. The skin was closed by nylon sutures. The total operation time was lo-15 minutes. The rats were housed in individual cages without special care and allowed to recover for 24 days from the surgery before next CSF sampling. At the time of sampling the rat was wrapped in a towel, the sampling cannula inserted to the upper end of the needle and CSF withdrawn by a slow aspiration. CSF (130 ~1) was withdrawn immediately after the surgery and every third to fourth day for 2-3 weeks.
A B
0.08 2 0.01 0.11 ?. 0.01
C D
0.08 i- 0.01 0.06 t 0.01
0.45 -i- 0.111 0.17 2 0.04t 0.15 t 0.09; 0.14 t O.Ol$
0.48 + 0.19 ;r. 0.14 k 0.14 2
0.06t 0.08*$ 0.06$ 0.02$
0.58 0.30 0.12 0.15
k 0.047 t 0.04t + 0.038 +_ 0.029
Mean value 2 SEM. *p
Brain Research Bulletin, Vol. 27, pp. 283-286. Q Pergamon Press plc, 1991. Printed in the U.S.A.
BRIEF COMMUNICATION
Changes in Physiological Parameters of Rat Cerebrospinal Fluid During Chronic Sampling: Evaluation of Two Sampling Methods IRENA WESTERGREN Department
of Neurology,
AND BARBRO
B. JOHANSSON
University Hospital, S-221 85 Lund, Sweden
Received
23 October
1990
WESTERGREN, I. AND B. B. JOHANSSON. Changes in physiological parameters of rat cerebrospinal fluid during chronic sampling: Evaluation of two sampling methods. BRAIN RES BULL 27(2) 283-286, 1991.-Although rat cerebrospinal fluid (CSF) is increasingly being used in pharmacological and biochemical research, methodological studies on basic physiological data are lacking. We have determined the albumin content and number of erythrocytes and leukocytes in CSF obtained by two different methods of sampling from cistema magna-repeated sampling from an implanted cannula and by repeated punctures. In the initial samples the albumin content was 0.08*0.03 pg/pl. Chronic cannulation of the cistema magna resulted in a meningeal reaction with increased cell and albumin content: a reaction that could be reduced but not prevented by using a sterile cannula. The number of leukocytes but not erythrocyteswas highly correlated to the albumin content. Repeated sampling in the absence of a permanent cannula did not significantly increase albumin content but carried a higher risk for erythrocyte contamination. CSF
Rat
Albumin
Erythrocytes
Leukocytes
(Hamilton Co. Inc., Whittier, CA 710). The tip of the needle perfectly fit the upper end of the cannula without leaving any dead space, thus the amount of CSF withdrawn could be controlled.
IN spite of the common use of rat cerebrospinal fluid in pharmacological studies, no data is available on the physiological levels of albumin and cell content, nor the possible effect of chronic cannulation on these parameters. Sampling of CSF from cistema magna in rat is usually not performed under sterile conditions. Following earlier described techniques (l-3, 6, 8-l l), we have in preliminary studies observed a considerable meningeal reaction. As a base for further studies on blood-CSF and brainCSF transfer under normal and pathological conditions, we have therefore determined the physiological baseline value for albumin and cells and to what extent these parameters can be altered during chronic cannulation or repeated samplings.
Implantation
of the Cannula and CSF Sampling
Male Sprague-Dawley rats (250-300 g body weight) were anesthetized with methohexital (Brietal) 60 mg/kg IP. The head was fixed in horizontal position in a stereotaxic apparatus, the incision area shaved and a l-cm midline incision was made in the skin to expose the dorsal and posterior surfaces of the skull. A hole was drilled with a small dental drill at the sag&al midline through the suture between the interparietal and occipital bone at 60” angle to the interparietal bone. So far the procedure was identical for all rats. The rats were divided into 2 groups.
METHOD
The tips of Venisystems Butterfly-21G steel needles (Abbot Scandinavia AB, 16421 Kista) were cut to a length of 7 mm from the point of insertion. The obturator from B-D spinal needles 90-7 (Becton, Dickinson and Company, Rutherford, NJ 07070) was cut to a length to make it just visible on the tip when inserted into the upper end of the needle. The lateral wings of the infusion set were reduced. The needle (Fig. 1A) was used as a chronically implanted cammla as well as for repeated cannulation. The cannula was either cleaned with physiological solution (nonsterile cannula) or sterilized by retaining it in 95% ethanol for 24 hours and flushed with sterile physiological solution before use. CSF was drawn with a lOO+l Hamilton needle
Rats With Indwelling Cannulae
Two additional holes were drilled in the interparietal bone for placement of screws. The obturator was placed into the cannula which was then inserted to its entire depth (i.e., 7 mm from the outer surface of the skull) into the prepared opening at approximately 60” angle to the interparietal bone by free hand. The position of the cannula in cistema magna (Fig. 1C) was controlled by removal of the obturator, insertion of the tip of the Hamilton needle and withdrawal of CSF. The cannula was then anchored
283
284
WESTERGREN
AND JOHANSSON
TABLE 1 ALBUMIN CONTENT IN RAT CISTERNAL FLUID @&I) DURING REPEATED SAMPLING WITH OR WITHOUT PERMANENT CANNULATION Experimental Group
Sample 1
2
3
J
-__
FIG. 1. The cannula tema magna (C).
(A), metal stop (B) and cannula’s
position in cis-
to the skull and to the screws with acrylic dental cement and closed with a metal stop (made by the local research technical department, Fig. 1B) placed in the upper end of the needle. The skin was closed by nylon sutures. The total operation time was lo-15 minutes. The rats were housed in individual cages without special care and allowed to recover for 24 days from the surgery before next CSF sampling. At the time of sampling the rat was wrapped in a towel, the sampling cannula inserted to the upper end of the needle and CSF withdrawn by a slow aspiration. CSF (130 ~1) was withdrawn immediately after the surgery and every third to fourth day for 2-3 weeks.
A B
0.08 2 0.01 0.11 ?. 0.01
C D
0.08 i- 0.01 0.06 t 0.01
0.45 -i- 0.111 0.17 2 0.04t 0.15 t 0.09; 0.14 t O.Ol$
0.48 + 0.19 ;r. 0.14 k 0.14 2
0.06t 0.08*$ 0.06$ 0.02$
0.58 0.30 0.12 0.15
k 0.047 t 0.04t + 0.038 +_ 0.029
Mean value 2 SEM. *p
