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The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, La Jolla, California 92037
Address all correspondence and requests for reprints to: Dr. Catherine L. Rivier, The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute, 10010 North Torrey Pines Road, La Jolla, California 92037. E-mail: crivier{at}salk.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several diverse technologies are presently used to inhibit neuropeptides; the most common are passive immunoneutralization, administration of receptor antagonists, treatment with antisense oligonucleotides, and gene knockout strategies. However, given that most neuropeptides are also widely distributed throughout peripheral tissues (e.g. immune system and gastrointestinal tract) (1, 2, 3), the demonstration of a neuropeptide’s CNS function requires a CNS-specific manipulation. As such, conventional gene knockout strategies (4) are inappropriate, and the use of targeted DNA recombination in vivo using adenoviruses (5, 6) is still in its infancy. Consequently, the majority of currently used methodologies rely on injection of agents either into the cerebroventricles (icv) or directly into brain tissue.
The use of antibodies to inhibit the actions of neuropeptides has been extensive and is associated with a number of advantages over other procedures. For example, of the numerous known neuropeptides, receptor antagonists for their cognate receptors have been developed for only a few, and even if available, they may be effective only at specific receptor subtypes. On the other hand, the restricted distribution, poor stability, and nonspecific toxicity of injected oligonucleotides have often made interpretation of in vivo antisense experiments difficult (7, 8). In contrast, antibodies do not necessarily require access to intracellular compartments to inhibit neuropeptide action and are extremely stable within biological tissues (9, 10, 11). Furthermore, antibodies administered icv have been shown to diffuse through brain tissue within a few hours of injection (12, 13, 14) and become concentrated at sites expressing the immunogenic epitopes (15). Given the relative ease of production, the high degree of specificity, and the wide availability of antisera/antibodies (10, 11), it is, therefore, not surprising that the use of intracerebral passive immunization procedures has been adopted by numerous workers (including ourselves) from many different fields of CNS investigation (16, 17, 18, 19, 20, 21, 22, 23).
The vast majority of investigators using icv passive immunization
procedures have made the assumption that the small quantities
(typically 1–10 µl) of antisera or purified antibodies administered
into the cerebroventricles inhibit the action of a neuropeptide
specifically within the brain and do not gain access to peripheral
tissues in substantial quantities. This assumption is probably based on
an understanding of limited transport of large mol wt proteins across
the blood-brain-barrier (24) and the unlikelihood that antibodies (160
kDa) can reach the systemic circulation in significant amounts after
icv administration. Consequently, most workers have not determined
whether the effects of antibodies/antisera administered icv could be
accounted for by actions within peripheral tissues (16, 17, 18, 19, 20, 21, 22). However,
the preceding article (25) describes experiments in which icv infusion
of 5 µl of a rabbit anti-tumor necrosis factor-
(TNF) antiserum
resulted in measurable amounts of corresponding rabbit anti-TNF
antibodies in jugular venous plasma. Furthermore, anti-TNF
administered icv 20 h earlier inhibited the actions of TNF in
systemic blood, suggesting that icv administration of antisera can
result in effectual concentrations of corresponding antibodies within
the peripheral circulation (25).
The purpose of the present work was 2-fold: 1) to define the temporal profiles of corresponding antibodies in peripheral blood after icv injection of several different neuropeptide antisera (sheep anti-CRF, rabbit anti-CRF, and rabbit anti-GnRH); and 2) to determine whether the levels of antibodies equivalent to those in peripheral plasma after icv injection of small amounts (5 µl) of neuropeptide antisera are sufficient to effectively neutralize the biological activity of their corresponding peptides using in vitro (rat anterior pituitary cells) and/or in vivo (iv CRF challenge) paradigms. To establish whether our observations were applicable to the use of icv immunization procedures in general, we used antisera from different host species, raised against two separate peptides, and of varying antibody titers.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals
Male Sprague-Dawley rats (initial body weight, 170–240 g) were
purchased from Harlan Sprague-Dawley Laboratories (Indianapolis, IN),
and housed in animal facilities adjacent to experimental rooms. They
were maintained on a 12-h light, 12-h dark cycle (lights on at
0600 h) and provided rat chow and water ad libitum. All
procedures described were approved by The Salk Institute animal use and
care committee.
Surgical preparation, antiserum administration, and blood
collection
Rats were equipped with cannulas for administration of antisera
via several different routes. Intracerebral guide cannulas (Plastics
One, Roanoke, VA) were stereotaxically implanted into the lateral
ventricle, third ventricle, or globus pallidus using stereotaxic
coordinates (nose bar, -3.3 mm) derived from Paxinos and Watson (26),
as described previously (27). Relative to Bregma, the coordinates used
were: lateral ventricle: anterior-posterior, -0.4 mm; lateral, -1.4
mm; dorsoventral, -3.5 mm; third ventricle: AP, -0.9 mm; lateral, 0.0
mm; dorsoventral, -8.0 mm; and globus pallidus: anterior-posterior,
-1.1 mm; lateral, -3.0 mm; dorsoventral, -6.5 mm. Seven to 9 days
later, intracerebral treatments (2–5 µl) were administered to freely
moving rats over a period of 1 min (infusion into cerebroventricle) or
3 min (into globus pallidus) via a connecting injection needle
(Plastics One) that extended 1 mm beyond the tip of the guide cannula.
Correct placement of guide cannulas was verified postmortum by infusion
of 5 µl India ink through the guide cannula assembly and visual
inspection of the ink’s distribution in approximately 1-mm sections of
chilled brain. Correct placement was defined as a distribution of ink
that was 1) throughout the ventricular system (i.e. third,
fourth, and lateral ventricles and cerebral aqueduct) in the case of
intraventricular guide implantation, or 2) confined to the globus
pallidus/surrounding tissue, but not within the ventricular system, in
the case of globus pallidus implantations.
Jugular venous cannulas were implanted 48 h before experimentation to permit blood sampling and iv injection (28). When ip injections were required, a cannula was inserted under the abdominal wall at the same time as iv surgery (29). Subcutaneous injections were made directly using a 27-gauge hypodermic needle. Within an experiment, the amount of antiserum injected via peripheral routes was the same as that injected via intracerebral routes. However, for the purposes of accuracy, antiserum that was administered peripherally was first diluted in 0.9% saline-0.1% BSA. The total injection volume was 0.5 ml via iv and ip routes and 0.2 ml via the sc route.
Blood was collected at various times after serum administration, and plasma was obtained by centrifugation (2000 x g). Plasma was stored at -20 C before analysis.
Titration of antiserum in plasma samples
Details of the antisera used for injection and the subsequent
reagents used in their titration in plasma are given in Table 1
. CRF and GnRH tracers were prepared by
radiolabeling rat/human CRF and mammalian GnRH with 125I by
the chloramine-T method, as described previously (30). Plasma samples
were serially diluted (1:2) in a buffer (30) containing 0.1
M NaCl, 0.05 M
Na2HPO4-NaH2PO4, 0.025
M EDTA, 0.1% azide (SPEA), and 0.1% crystalline BSA-0.1%
Triton-X (SPEAB) to final volumes of 100 µl and incubated overnight
at room temperature with 50 µl radiolabeled peptide in SPEAB. The
following day, samples were incubated for 2 h at room temperature
with 50 µl 1:20 diluted secondary antiserum and 200 µl 10%
polyethylene glycol in SPEAB-Triton X. Samples were then washed with
500 µl SPEA and centrifuged at 2000 x g for 45 min,
the supernatants were decanted, and the dried pellets were counted in a
-counter. The percentage of tracer bound was calculated after
accounting for nonspecific binding (no plasma present), and the plasma
titer of each sample was calculated by determining the dilution of
plasma that bound 50% of tracer (using Figure Perfect Software,
Biosoft, Ferguson, MO). In each experiment, the titers of the undiluted
antisera employed were also determined in parallel (see Table 1).
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Hormone measurements
ACTH concentrations of conditioned tissue culture medium
(diluted 1:14 in SPEAB) and plasma samples (undiluted) were measured
using a commercial immunoradiometric assay (Allegro, Nichols Institute,
San Juan Capistrano, CA) (31). In these experiments, within- and
between-assay coefficients of variation were 9% and 7%, respectively,
at 0.04 ng/ml and 2% and 4% at 0.38 ng/ml.
LH concentrations of conditioned tissue culture medium (diluted 1:24 in SPEAB) were measured by a double antibody RIA using standard (RP-3) provided by the NIDDK and a primary antibody (no. 15) provided by Dr. G. Niswender (Fort Collins, CO). Precipitation of antibody was accomplished with the addition of sheep anti-rabbit IgG followed by a wash (0.5% Tween-20 and 5% polyethylene glycol) and centrifugation. The within- and between-assay coefficients of variation of this assay were 8% and 21%, respectively, at 0.6 ng/ml and 6% and 18% at 4.3 ng/ml.
Data presentation and statistical analyses
The data are presented as the mean ± SEM, and
the numbers of subjects in each experimental group are indicated in the
figure legends. Statistical analyses were performed using either ANOVA
with repeated measures, with least squared means as a
post-hoc test, or unpaired Student’s t test. A
two-tailed probability of less than 5% (i.e..
P < 0.05) was considered statistically
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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5 µl), we first constructed
[125I]CRF titration curves of plasma samples from rats
treated iv with 1 or 5 µl sheep anti-CRF and compared them to the
titration curve produced by the uninjected antiserum (see Fig. 1
). Plasma from animals treated iv with
NRS displayed no specific binding of [125I]CRF (data not
shown). In contrast, serial dilutions of plasma from rats injected iv
with either 1 or 5 µl sheep anti-CRF 1 h earlier produced
titration curves that were parallel to one another as well as to that
of the uninjected antiserum (see Fig. 1). Calculation of the dilutions
of samples that produced 50% specific binding yielded the titer of
each test sample. The plasma of animals injected with 1 µl antiserum
had a 4.73-fold lower titer than that from animals injected with 5 µl
antiserum, indicating the high degree of precision of this procedure.
Comparison of the plasma titers with those of the antiserum alone
indicated that the antiserum was diluted by 15,000- and 3,170-fold in
animals injected with 1 and 5 µl, respectively. Serial dilutions of plasma from rats treated iv with either 5 µl rabbit anti-CRF (rC69) or rabbit anti-GnRH (L45) also showed parallel displacement curves compared with those of the respective antiserum alone (data not shown). Specific binding of either [125I]CRF or [125I]GnRH by even the least dilute of jugular venous plasma samples collected from control animals [untreated or injected (iv or icv) with normal serum] was always less than 5%.
Antiserum infused icv produces a rapid appearance and sustained
presence of corresponding antibodies in systemic venous blood
To determine whether significant amounts of antibodies are present
in systemic blood after icv infusion of antiserum, we compared the
binding of [125I]CRF by jugular venous plasma from rats
infused icv with 5 µl sheep anti-CRF to that by plasma from rats
treated iv with the same amount of antiserum. Serial dilutions of
plasma exhibited substantial binding of [125I]CRF as
early as 30 min after administration icv (into the right lateral
cerebroventricle; see Fig. 2A). The
titration curve generated from these samples was parallel that obtained
with plasma samples from rats treated with antiserum iv (into the
jugular vein; Fig. 2A). However, the icv titration curve was markedly
(42-fold) shifted to the left of the iv titration curve, indicating
that corresponding antibody levels in venous plasma 30 min after icv
infusion of antiserum were 2.4% of those 30 min after iv injection of
antiserum. The displacement of icv and iv titration curves became less
pronounced with time (see Fig. 2B), such that by 8 h these curves
were virtually superimposable (Fig. 2C), indicating that by this time
the systemic plasma levels of anti-CRF antibodies after icv and iv
administration were similar.
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and Table 1). As expected,
plasma antibody levels were highest at the earliest time point after iv
administration of antiserum (30 min) and declined slowly, such that
levels at 24 h were 40–53% of those at 30 min. In contrast, only
very low, but still detectable, levels of antibodies were apparent in
systemic plasma 30 min after icv infusion of antisera (2–6% of levels
30 min after iv antiserum administration). Systemic plasma levels of
corresponding antibodies after icv antiserum administration increased
steadily thereafter, such that by 2 and 4 h after antiserum
infusion, levels were 46–53% and 68–76% of those after iv antiserum
treatment, respectively. Eight and 24 h after antisera
administration, plasma levels of corresponding antibodies produced by
iv and icv administration of antisera were not significantly different
(by ANOVA with repeated measures, least squared means).
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). The profile of anti-CRF antibodies in
systemic plasma after ip administration of 5 µl of anti-CRF antiserum
differed markedly from that produced by the iv route of administration.
Levels increased slowly after ip antisera administration and only
achieved levels similar to those produced by iv injection at 8 and
24 h. Indeed, although 30 min after ip antiserum administration,
plasma antibody levels (titer, 25.2 ± 3.2) were far greater than
those after icv antiserum treatment (titer, 2.8 ± 0.3), plasma
levels at the remaining time points were very similar between these two
routes of administration (Fig. 4A). On the other hand, sc injection of
anti-CRF did not result in readily measurable levels of anti-CRF
antibodies in systemic plasma until 8 h (Fig. 4A).
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). In contrast, infusion directly
into brain tissue at a site distant from cerebroventricles (globus
pallidus) produced levels of anti-CRF antibodies in systemic plasma
that were not measurable until 4 h. Although they increased
thereafter, levels in systemic plasma 24 h after globus pallidus
infusion of antiserum only reached 45% of those produced by
administration into the lateral ventricle, the third ventricle, or the
general circulation.
Intracerebroventricular antisera produces concentrations of
corresponding antibodies in peripheral blood that are sufficient to
inhibit neuropeptide action in vitro and in vivo
An in vitro experimental paradigm was used to determine
whether the concentrations of antibodies equivalent to those achieved
in systemic plasma after icv antisera administration were capable of
immunoneutralizing their corresponding peptides. This involved testing
the effectiveness of relevant dilutions of anti-CRF or anti-GnRH
antiserum to inhibit either CRF-induced ACTH secretion or
GnRH-stimulated LH secretion from primary cultures of rat anterior
pituitary cells. Relevant dilutions were determined by calculating the
dilution of antiserum in systemic blood 1–24 h after its icv
administration (5 µl; see Fig. 1 for explanation of calculations).
The final mean dilution was derived from the average of two experiments
employing sheep anti-CRF (Figs. 3 and 4A) and one each using rabbit
anti-CRF and rabbit anti-GnRH (Fig. 3). This yielded values that were
remarkably consistent between the different experiments and antisera
(see Table 2) and varied between
approximately 1:15,000 at 1 h and 1:5,000 at 8 h, with other
time points yielding intermediate values (see Table 2). We chose the
lowest (1:5,000) and highest (1:15,000) dilutions of antisera to test
in our rat anterior pituitary cell bioassays.
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).
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). Indeed at
a 1:5,000 dilution of either sheep anti-CRF or rabbit anti-CRF, ACTH
secretion induced by the two lower doses of CRF (0.05–0.5
nM) was completely abolished (Fig. 5). However, only the
two anti-CRF antisera significantly inhibited peptide-induced hormone
secretion at a dilution of 1:15,000. The efficacy of these three
antisera at inhibiting peptide-induced hormone secretion from the
anterior pituitary cells corresponded to the relative titers of each
antiserum (see Table 1 and Fig. 5).
As well as testing whether the levels of antibodies in systemic
blood after icv administration of antisera were sufficient to inhibit
neuropeptide biological activity in vitro, we also examined
the in vivo impact of prior icv administration of sheep
anti-CRF antiserum on a well characterized peripheral effect of CRF
(pituitary ACTH secretion). Rats were pretreated with 5 µl of either
sheep anti-CRF or NSS administered into the right lateral
cerebroventricle (icv). Three and a half to 4 h later, plasma ACTH
concentrations in NSS- and anti-CRF-treated rats were similar (40–60
pg/ml; see Fig. 6). At 4 h after icv
pretreatments, a single bolus of CRF (0.3 nmol/kg) was administered iv.
In rats pretreated with only NSS (icv), CRF produced a rapid rise (peak
at 10 min, 276 ± 23 pg/ml) in plasma ACTH concentrations that was
sustained for 60 min after CRF injection (Fig. 6). Prior administration
of sheep anti-CRF (5 µl, icv) markedly reduced the plasma ACTH
response to CRF (Fig. 6). Integration of the plasma ACTH concentrations
(minus basal levels) over the 60-min period after CRF injection
indicated that pretreatment icv with anti-CRF inhibited the plasma ACTH
response to CRF by 88% (NSS icv, 141 ± 26 pg/ml·h; anti-CRF,
17 ± 12 pg/ml·h; P < 0.001, by Student’s
unpaired t test).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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These studies also show that amounts of antisera in the range typically used in icv immunoneutralization studies (5 µl) rapidly (within 1–4 h) produce systemic plasma levels of corresponding antibodies that when used in in vitro paradigms are sufficient to inhibit the biological activity of their corresponding peptide. This was apparent for three different antisera of markedly different antibody titers, indicating that this finding is not peculiar to a particularly high titer antiserum and may be manifest with a wide range of antisera used for immunoneutralization studies. We also found that the in vivo administration of anti-CRF icv (5 µl) virtually abolished the plasma ACTH response to an iv CRF challenge 4 h later. As the primary site of CRF action in elevating plasma ACTH concentrations is the anterior pituitary gland, this indicates that the sheep anti-CRF administered icv neutralized CRF by an action within the periphery. Moreover, recent studies showing that CRF administered iv does not readily penetrate the blood-brain barrier (34) reinforce the view that the inhibitory effect of anti-CRF administered icv could not be accounted for by interactions between CRF and CRF antibodies within the CNS. Together with the data presented in the previous article (25), these results provide compelling evidence that icv administration of small amounts (5 µl) of antisera/antibodies can produce immunoneutralization of peptides in peripheral tissues.
This finding has important implications for our interpretation of previous experimental data as well as for future experimental design. Passive immunoneutralization via the icv route has been an extremely common method to investigate the function of neuropeptides within the CNS (16, 17, 18, 19, 20, 35, 36, 37, 38, 39, 40, 41, 42, 43). Moreover, administration of either antisera or purified antibodies into the cerebroventricles has been used to study the CNS functions of cytokines (44), growth factors (22), eicosanoids (45), steroids (46), and their receptors (47, 48). Such works have investigated the biological roles of these factors within the CNS either under basal conditions or in response to diverse stimuli of both CNS (e.g. cognitive challenge and brain damage) and peripheral (e.g. manipulations of blood volume and immune stimulation) origin and have contributed greatly to our present understanding of their CNS activities. However, by far the majority of studies (65 of 68 articles we have reviewed) have not included parallel experiments determining the effects of administration of the same antiserum or antibody via a peripheral route and, therefore, cannot discount the possibility that the effects of icv antiserum was actually manifest within peripheral blood or tissues. Clearly, the data we present here indicate that concluding that the effect of a particular antiserum/antibody infused icv is due to an action within the brain requires substantiating evidence. The antiserum’s concentration (or titer) in the periphery should be assessed, and the effect of similar blood levels in the absence of markedly elevated brain antibody concentrations should be determined. In this regard, the ip injection of an antiserum would appear to provide an appropriate control. The present work also suggests that even when antisera are administered directly into brain tissue, peripheral control experiments are required. We chose the globus pallidus as an injection site because of its relatively greater distance from cerebroventricles than most other regions of the brain. Even after intraglobus pallidus administration of 2 µl antiserum, readily detectable levels of the corresponding antibodies were apparent in systemic plasma by 4 h. It should be noted that antiserum administered into a brain site far closer to the ventricular system, such as the paraventricular nucleus of the hypothalamus, is likely to produce a plasma profile of corresponding antibodies that is intermediate between those described here for the icv and globus pallidus routes of administration.
The exit route of antibodies out of the CNS and into systemic blood was not investigated. However, several studies have shown that other large proteins, such as albumin (70 kDa) (49), ovalbumin (44 kDa) (50), horseradish peroxidase (40 kDa) (51), and wheat-germ agglutinin (35 kDa) (51) are present within peripheral blood or tissues within hours of their infusion into either cerebrospinal fluid (CSF) or specific brain sites. The major means by which albumin leaves the CNS has been discussed (52) and involves drainage into regional lymph nodes via prolongations of the subarachnoid space along cranial nerves (49). Whether the same mechanism accounts for the dissipation of antibodies from CSF or brain tissue is not known. However, given the relatively slow rate of exit of antibodies compared with the rate of exit by active transport mechanisms (half-time disappearances from CSF of <30 min) (34, 53), it seems probable that, like albumin, antibodies exit the brain largely via passive readsorption of CSF (54).
It is pertinent to consider the use of the icv route of administration
of agents other than antisera. It seems reasonable to assume that if
molecules as large as antibodies (160 kDa) readily reach the systemic
circulation after their icv administration, so, too, do other
molecules. In support of this conclusion, the appearance of a number of
peptides in systemic blood after their icv administration has been
reported (34, 53, 55, 56, 57, 58), and in some cases active transport
mechanisms have been proposed (34, 53, 57). We should, therefore,
consider whether the effect of a substance administered icv is
accounted for by an action in brain or by an action in peripheral
tissues after transport out of the CNS. It is generally accepted that
to ascribe the action of a peptide to a CNS activity, it must elicit
responses after icv administration of doses lower than those required
to produce the same effect when administered peripherally. However,
most peptides have an extremely short t1/2 in blood; for
example, TNF- has a t1/2 of less than 10 min (55).
Consequently, an iv bolus of peptide results in a marked, but
short-lived, elevation in its plasma concentration. In contrast, the
icv injection of peptide, should it exit to blood in a manner akin to
that of antibodies, will result in a sustained elevation in the plasma
peptide concentration due to the relatively slow dissipation from CSF
to systemic blood. Indeed, by 30 min after TNF- administration,
trunk blood levels of TNF- are actually higher after icv infusion
than after iv injection (55). Therefore, it remains possible that the
prolonged exposure of peripheral tissues to elevated concentrations of
peptides could account for some of the documented effects of peptides
administered icv, even in cases where the icv route of administration
is reported to be more potent than peripheral injections. Collectively,
the present work and studies described herein argue that the use of the
icv route of administration of reagents to investigate CNS activities
requires more carefully controlled studies than are generally performed
at present. Furthermore, whether transport of endogenous biological
molecules from the CSF to the bloodstream represents a mechanism by
which the brain can influence peripheral targets should also be
considered (56).
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Acknowledgments |
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Footnotes |
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2 Present address: North Western Injury Research Centre, Stopford
Building, University of Manchester, Oxford Road, Manchester, United
Kingdom M13 9PT.
3 Investigator with The Clayton Foundation.
Received June 25, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(TNF-) action within the central nervous
system markedly reduces the plasma adrenocorticotropin response to
peripheral local inflammation in rats. J Neurosci 17:3262–3273 on the
plasma ACTH response to lipopolysaccharide in rats. Endocrinology 139:000–000
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