Comparison Among Different Approaches for Sampling Cerebrospinal Fluid in Rats

doi:10.1016/S0361-9230(96)00176-1

Brain Research Bulletin

Volume 41, Issue 5, 1996, Pages 273-279

https://doi.org/10.1016/S0361-9230(96)00176-1 Get rights and content

Abstract

Two approaches for time-resolved sampling of cerebrospinal fluid (CSF) in rats were compared regarding performance, reproducibility, and extent of the inevitable trauma caused by the implantation of a sampling tube. Several parameters were checked to evaluate the injury: blood cell contamination of CSF; concentrations in CSF of the cytosolic proteins neuron-specific enolase (NSE) and S-100 (chiefly present in astrocytes); blood-brain barrier leakage of a dye-protein complex; viability of nervous tissue cells as assessed by dye exclusion; light and electron microscopy. In one sampling method, a tube was forced epidurally into the cisterna magna via a hole in the calvarium, consistently damaging the meninges and the nervous tissue. When using the alternative sampling method, the tube was instead affixed to the posterior atlanto-occipital membrane and connected with the cisterna magna via a hole in the membrane. Such a procedure caused negligible damage. Both techniques induced an inflammatory response. We advocate the use of the second approach, i.e., to sample CSF via a hole in the atlanto-occipital membrane, as the method of choice due to its high reproducibility. It is fairly rapid, and associated with a negligible injury. Copyright © 1996 Elsevier Science Inc.

Section snippets

INTRODUCTION

Time-resolved sampling of cerebrospinal fluid (CSF) enables evaluation of dynamic biochemical and pharmacological events in the extracellular milieu of the central nervous system (CNS) 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 18, 19, 20, 21, 23. The preferred origin of CSF in rats is the cisterna magna. It is readily available and has a large volume (≈ 190 μl; cistern and adjacent subarachnoid spaces ≈ 246 μl; one lateral ventricle just ≈ 10 μl) [3]. The techniques in use require surgical insertion of

Animals

Adult outbred Sprague-Dawley rats were used (n = 360; body weight ≈ 300 g; B and K Labs. AB, Stockholm, Sweden). The light was on between 6 a.m. and 6 p.m. The animals had free access to pelleted food and water. Permission to the experiments was granted by the Animal Experiments Ethical Committee (authorizations O $29393$ , O $7095$ , and A $6092$ ).

Anesthesia and Surgery

One group of animals was anaesthetized during the surgery and the CSF sampling either by intraperitoneal injection of a mixture of pentobarbital sodium (60

General Comments

Both surgical procedures could be completed by the surgeon in 1 h, including the time required for the polymerization of the acrylic cement. Based on the records for 193 rats, ≥ 65% of the tubes implanted according to surgical technique I were patent after one day. Surgical techniques II had a patency rate ≥ 90% after one day (n = 128).

The tightness of the tube was checked by dissection of the extracranial tissue 2 h after the surgery in animals with EBA infused into the cisterna magna at the

DISCUSSION

A major problem in experiments with time-resolved sampling of CSF is the damage caused by the surgery necessary for the implantation of the sampling tube. The commonly used technique, surgical technique I, invariably damaged the meaninges and the adjacent nervous tissue. These results are in agreement with those published previously 4, 7, 9, 19, 20, 21, 23. In contrast, surgical technique II caused negligible evidence of trauma, restricted to the immediate vicinity of the tube and hole, or none

Acknowledgements

We thank Mr. K. Hansson, Mr. T. Jönsson, Ms. I.-L. Larsson, Ms. E. Malm, Ms. E. Mattsson, Ms. M. Miikiver, and Mr. M. Vona for their expert assistance and Mr. T. Seeman, MD, PhD, for valuable help.

References (27)

H.J. Bouman et al.
A rapid and simple technique for multiple sampling of cerebrospinal fluid in freely moving rats
Brain Res. Bull.
(1979)
S. Frankmann
A technique for repeated sampling of CSF from the anesthetized rat
Physiol. Behav.
(1986)
A. Grasso et al.
Localization of 14-3-2 protein in the rat brain by immuno electron microscopy
Brain Res.
(1977)
R.H. Hatfield et al.
CSF neuron-specific enolase as a quantitative marker of neuronal damage in a rat stroke model
Brain Res.
(1992)
Y.-L. Huang et al.
A new approach for multiple sampling of cisternal cerebrospinal fluid in rodents with minimal trauma and inflammation
J. Neurosci. Methods
(1995)
M.E. Kornhuber et al.
A method for repeated CSF sampling in freely moving rat
J. Neurosci. Methods
(1986)
M.E. Kornhuber et al.
Positive correlation between contamination by blood and amino acid levels in cerebrospinal fluid of the rat
Neurosci. Lett.
(1986)
B.W. Moore
Chemistry and biology of two proteins, S-100 and 14.3.2, specific to the nervous system
Int. Rev. Neurobiol.
(1972)
L. Persson et al.
14-3-2 protein in rat brain
J. Neurol. Sci.
(1978)
I. Westergren et al.
Changes in physiological parameters of rat cerebrospinal fluid during chronic sampling: Evaluation of two sampling methods
Brain Res. Bull.
(1991)

Q. Yang et al.

S-100 β has a neuronal localisation in the rat hindbrain revealed by an antigen retrieval method

Brain Res.

(1995)

R. Bakay et al.

Pathophysiology of cerebrospinal fluid in head injury: Part 2. Biochemical markers for central nervous system trauma

Neurosurg.

(1986)

D. Burns et al.

A kinetic analyses of 5-hydroxyindoleacetic acid excretion from rat brain and CSF

Biol. Psych.

(1976)

Cited by (19)

An applicable method of drawing cerebrospinal fluid in rats
2016, Journal of Chemical Neuroanatomy
Component analysis of cerebrospinal fluid (CSF) is frequently required to probe the causes and pathologic mechanisms of disease and effective drugs in experimental studies of the central nervous system. Rat and mouse are two kinds of most frequently used animals in experimental studies. Rats are considered to be the most suitable animal for experimental analysis of CSF both on cost and manipulability as mice are too small for drawing CSF. However, drawing CSF from rats is still not easy, which makes many researchers choose bigger animals, such as rabbits. This paper introduced a highly applicable technique of CSF collection from cerebellomedullary cistern (CC) in rats.
CSF collection with this technique was performed by direct CC puncture using a collection apparatus with negative pressure. The apparatus consists of a 1 ml syringe, a disposable intravenous infusion needle and a clip. The needle was cut and made less sharp than the original one to avoid injury to the brain and spinal cord.
We have collected CSF multiple times from each rat with this approach and the collection lasted less than 30 s each time on average. The length of the collection needles of the CSF was conformed to the different body sizes (weight) of the rats in the 3 groups. Compared with currently existing methods, this is faster, safer, simpler and repeatable.
CSF collection by CC puncture using a negative pressure collection apparatus is fast to operate, safe to the rats, and maximum amount of CSF can be collected.
High performance collection of cerebrospinal fluid in rats: Evaluation of erythropoietin penetration after osmotic opening of the blood-brain barrier
2013, Journal of Neuroscience Methods
An important issue to be considered when studying a new drug for treatment of central nervous system (CNS) diseases is its ability to cross the blood–brain barrier (BBB) and distribute throughout the brain. As cerebrospinal fluid (CSF) has demonstrated to be an invaluable reservoir to study CNS availability of therapeutic proteins, we have developed an improved method for CSF sampling from the cisterna magna of rats. The technique enables the simple and rapid collection of adequate quantities (50–75 μl) of blood-free CSF, rendering a high percentage of animal survival (99%) without clinic or neurological consequences. Its success in avoiding blood contamination of CSF lays in the use of a mixture of lidocaine/ephinephrine topically injected in the rat's suboccipital area and neck. Another relevant feature of the methodology is its low cost, since the puncture device can be easily assembled with cheap and available materials and, more importantly, neither expensive stereotaxic equipment nor frame is required. The present method is demonstrated by studying the CSF pharmacokinetics of recombinant human erythropoietin (rhEPO), a well-studied therapeutic candidate for neurological diseases. Moreover, we applied this technique to evaluate a strategy of osmotic disruption of the BBB to achieve a faster delivery of rhEPO into the CNS.
The comparison of three methods of drawing cerebrospinal fluid in rabbit
2012, Journal of Neuroscience Methods
Citation Excerpt :
Since CSF drawing is very frequently used procedure, what is the best method has raised our concern. In this study, we performed and compared the three common ways: skull drilling (Huang et al., 1996), lumbar puncture (Wang et al., 2005; De la Calle and Paíno, 2002) and atlanto-occipital membrane puncture (Haslberger and Gaab, 1986; Vistelle et al., 1989; Huang et al., 1996), in order to find an ideal one that is the simplest, fastest, and with the highest repeatability. Human lumbar puncture needles (#4, Bangcheng Co., Shanghai, China) with about 0.4 mm outside diameter: they were cut to be 4 cm in length and sharpened under the help of microscope with a grindstone (#1200), the tips were made less sharp than the original ones (see the part of discussion and Fig. 3); crew cut bone drills (RA-II, Yancheng Co., China, 8 mm outside diameter): to avoid easily break the cerebral dura mater, the tip of the drill were blunted a little using #200 first and then #1200 grindstones; silicone tubes with 1.5 mm inside diameter and 2 mm outside diameter; suture needles; sutures; alcohol swabs; dry cotton balls; scissors, forceps; animal fixing frame; 1 ml, 5 ml and 20 ml syringes; #5 and #7 syringe needles; trays; 1% of pentobarbital sodium.
Component analysis of cerebrospinal fluid (CSF) is frequently required to probe the cause of disease, pathologic mechanisms and effective drugs in the experimental study of central nervous system. An ideal way of drawing CFS is very important for the successful analysis. However, pros and cons of types of CSF drawing have never been analyzed and compared. The purpose of this study was to choose an ideal method of obtaining CSF in rabbits by comparing the three usual ones: skull drilling, lumbar puncture and atlanto-occipital membrane puncture. In the study, the rabbits were randomly divided into three groups: skull drilling group, lumbar puncture group and atlanto-occipital membrane puncture group, and our modified puncture needles were used. The rates of success, operating time and repeatability, etc. of the three methods were compared. The results suggest that the atlanto-occipital membrane puncture method is the best. The method was the simplest, fastest, and most repeatable of the three, furthermore, with this method, the most CSF was drawn and achieved the highest success rate.
Cisterna magna cannulated repeated CSF sampling rat model - effects of a gamma-secretase inhibitor on Aβ levels
2012, Journal of Neuroscience Methods
Citation Excerpt :
The transcutaneous approach includes both direct needle access (Takasugi et al., 2005; Nirogi et al., 2010) or open surgical access to the cisterna magna in the back of the neck (Pegg et al., 2010). In contrast, surgical trans-occipital bone approach (Sarna et al., 1983; Consiglio and Lucion, 2000) or catheterization via the atlanto-occipital membrane in the back of the neck (Huang et al., 1996) has been used to permanently implant catheters into the cisterna magna to enable repeated CSF sampling. Some reports have also used an indwelling cannula in the lateral ventricle to sample CSF over time via parietal bone access (Cassar et al., 2010).
Cerebrospinal fluid (CSF) provides a window into central nervous system (CNS) physiology and pathophysiology in human neurodegenerative conditions such as Alzheimer's disease. Changes in CSF bioanalytes also provide a direct readout of target engagement in the CNS following pharmacological interventions in clinical trials. Given the importance of tracking CNS bioanalytes in drug discovery, we have developed a novel cisterna magna cannulated rat model for repeated CSF sampling and used it to assess an amyloid beta (Aβ) lowering agent. The surgically implanted cisterna magna cannula was patent over a period of 1–2 weeks and enabled repeated sampling of CSF (volume of ∼30–50 μL/sample) from each rat. CSF Aβ40 levels showed good intra-animal variability across time points and inter-animal variability within a time point. Peripheral treatment with a gamma-secretase inhibitor (GSI) led to a rapid and robust decline in CSF Aβ40 levels that returned to baseline over 24–96 h after dosing. Terminal brain, CSF and plasma Aβ levels measured at 24 h after dosing demonstrated robust Aβ lowering and showed excellent correlation across these compartments. These results are the first pharmacological validation of the repeated CSF sampling rat model for Aβ lowering agents. This model can have broad applicability in pharmacological evaluation for diverse CNS targets.
Elevating intracranial pressure reverses the decrease in deoxygenated hemoglobin and abolishes the post-stimulus overshoot upon somatosensory activation in rats
2010, NeuroImage
BOLD fMRI localizes activated brain areas by measuring decreases of deoxygenated hemoglobin (deoxy-Hb) caused by neurovascular coupling. To date, it is unclear whether intracranial pressure (ICP) modifies deoxy-Hb signaling for brain mapping. In addition, ICP elevation can test whether the BOLD post-stimulus undershoot, a transient hypo-oxygenation following functional activation, is due to vascular compliance rather than elevated cerebral metabolic rate of oxygen (CMRO₂). We addressed these questions by studying the effect of ICP elevation on neurovascular coupling. In anesthetized rats, a cranial window was implanted over the somatosensory cortex. Using laser Doppler flowmetry and optical spectroscopy, changes in cerebral blood flow (CBF), cerebral blood volume (CBV) and deoxy-Hb were measured during electrical forepaw stimulation. Neuronal activity was monitored by somatosensory evoked potentials. ICP was elevated by subarachnoideal and intracisternal infusion of artificial cerebrospinal fluid. ICP elevation did not abrogate neurovascular coupling. However, the concomitant deoxy-Hb decrease was reduced (ICP = 14 mmHg) and reversed (ICP = 28 mmHg). Therefore, the validity of BOLD fMRI has to be questioned during increased ICP. Moreover, the amplitude of the deoxy-Hb post-stimulus overshoot was reduced with ICP elevation. CMRO₂ was not elevated during the post-stimulus response. Therefore, these data provide experimental evidence that the BOLD post-stimulus undershoot is a passive vascular phenomenon.
Comparing levels of biochemical markers in CSF from cannulated and non-cannulated rats
2010, Journal of Neuroscience Methods
Citation Excerpt :
Overall, these results suggest that levels of some biomarkers may be altered in CSF from cannulated versus non-cannulated rats. In addition to the two collection methods examined here, alternative techniques, cannulated and non-cannulated, are used by others (Huang et al., 1996; Pegg et al., 2010). Each method of collection may affect CSF biomarkers distinctly.
Cerebrospinal fluid (CSF) is commonly used for assessing biomarkers of drug efficacy or disease progression in the central nervous system. Studies of CSF from pre-clinical species can characterize biomarkers for use in clinical trials. However, obtaining CSF from pre-clinical species, particularly rodents, can be challenging due to small body sizes, and consequently, low volumes of CSF. Surgical cannulation of rats is commonly used to allow for CSF withdrawal from the cisterna magna. However, cannulae do not remain patent over multiple days, making chronic studies on the same rats difficult. Moreover, CSF biomarkers may be affected by cannulation. Thus cannulation may contribute confounding factors to the understanding of CSF biomarkers. To determine the potential impact on biomarkers, CSF was analyzed from cannulated rats, surgically implanted with catheters as well as from non-cannulated rats. Brain protein biomarkers (αII-spectrin SBDP150 and total tau) and albumin, were measured in the CSF using ELISA assays. Overall, cannulated rat CSF had elevated levels of the biomarkers examined compared to non-cannulated rat CSF. Additionally, the variation in biomarker levels observed among CSF from cannulated rats was greater than that observed for non-cannulated rat CSF. These results demonstrate that in some cases, biomarker assessment using CSF from cannulated rats may differ from that of non-cannulated animals and may contribute confounding factors to biomarker measurements and assay development for clinical use.

View all citing articles on Scopus

View full text

Comparison Among Different Approaches for Sampling Cerebrospinal Fluid in Rats

Abstract

Section snippets

INTRODUCTION

Animals

Anesthesia and Surgery

General Comments

DISCUSSION

Acknowledgements

Brain Res. Bull.

Physiol. Behav.

Brain Res.

Brain Res.

J. Neurosci. Methods

J. Neurosci. Methods

Neurosci. Lett.

Int. Rev. Neurobiol.

J. Neurol. Sci.

Brain Res. Bull.

Brain Res.

Pathophysiology of cerebrospinal fluid in head injury: Part 2. Biochemical markers for central nervous system trauma

Neurosurg.

A kinetic analyses of 5-hydroxyindoleacetic acid excretion from rat brain and CSF

Biol. Psych.