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Department of Medicine (H.J.W., A.L.), University of Birmingham, Birmingham B15 2TT, United Kingdom; Department of Anatomy and Cell Biology (M.B.), UMDS, Guy’s Hospital, London SE1 9RT, United Kingdom; Department of Medicine, Physiology and Paediatrics (D.J.H.), Lawson Research Institute, London, Ontario N6A 4V2, Canada; Department of Surgery (S.C.-H., J.M.P.H.), University of Bristol, Bristol, BS2 8HW, United Kingdom
Address all correspondence and requests for reprints to: Ann Logan, Department of Medicine, The University of Birmingham, Wolfson Research Laboratories, Queen Elizabeth Medical Centre, Edgbaston, Birmingham, B15 2TH, United Kingdom. E-mail: a.logan{at}bham.ac.uk
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In the CNS, IGF-II messenger RNA (mRNA) is detected from midgestation onwards (3), with significant levels of expression maintained in the leptomeninges, choroid plexus, and microvasculature after maturity, correlating with the sustained synthesis of immunoreactive IGF-II (4, 5). The peptide also becomes sequestered distal to these sites of synthesis within the myelin sheaths of individual axons and in nerve tracts (4). The significance of the distal depots of IGF-II peptide remains to be established; however, the spatial disparity between sites of synthesis and localization suggests that an effective mechanism of IGF-II translocation is operational. The CSF is possibly one transport route because, subsequent to secretion by the choroid plexus, it circulates through the ventricles and ultimately into the subarachnoid space and its perivascular extensions, thereby infusing the whole brain (6). Samples of CSF from the subarachnoid space of intact mature brains contain IGF-II (7), implying that circulating CSF distributes IGF-II peptide, secreted from mesenchymal support structures, around the brain in a manner analogous to that of endocrine trophins. We suggest that just as hormones are secreted from a glandular epithelium into the systemic circulation, to affect receptor-bearing target cells distally, IGF-II is released from the choroid epithelium, into the CSF, and is then circulated to depots or distal targets.
IGF transport and bioactivity are regulated by a family of six high affinity binding proteins (IGFBP-1–6), which are complexed to the IGFs within the circulation and throughout the extracellular space [see reviews by Clemmons (8) and Jones and Clemmons (9)]. Within the intact CNS, IGFBP-2 and IGFBP-4, -5, and -6 mRNA expression have been demonstrated in the choroid plexus and meninges of the ventricular system, with IGFBP-2 predominating (4, 10, 11, 12, 13, 14). Small quantities of IGFBP-1 peptide have also been identified within the intact brain (10, 15). Under pathological conditions, IGFBP-2, IGFBP-3, and IGFBP-6 have been detected in CSF (16, 17, 18, 19), suggesting that IGFBPs are involved in IGF-II transport from ventricular sites of synthesis to sites of storage and/or bioactivity.
IGF-II binds to specific, high affinity receptors (IGF-IR and IGF-IIR) that are expressed by a diverse range of cell types. IGF-IR mRNA is widely distributed in the CNS from early development and levels decline postnatally, although residual expression in (a) neurons of defined regions of gray matter (20, 21, 22, 23); (b) glia of the white matter of the cerebral and cerebellar hemispheres, and brain stem, (24); (c) epithelial cells of the choroid plexi (10, 25, 26, 27); (d) ependyma of the third ventricle (10); and (e) brain capillary endothelial cells (10, 28) remains high. It is evident that most IGF-II activity is mediated via IGF-IR and its tyrosine kinase signalling pathway (29, 30). The suggestion that IGF-IIR may also mediate some actions through signaling pathways other than tyrosine kinases (31, 32) has not been substantiated. Nevertheless, IGF-IIR has a widespread distribution in the developing CNS (33) but, in the adult, is confined to specific neuronal perikarya, for instance, those of the pyramidal cell layer of the hippocampus and the granule cell layers of the dentate gyrus and cerebellar cortex (34, 35). Lower levels are also observed in the choroid plexus and meninges (34). By contrast to the distribution of detectable levels IGF-IIR mRNA, almost all neurons and astrocytes of the brain are IGF-IIR immunopositive (23, 36). While it is possible that these cells are expressing undetectable mRNA levels, it is also possible that the immunoreactivity detected represents soluble forms of the receptor that have been shed from distal sites of synthesis.
IGFs have neurotropic (37, 38) and gliogenic (39) activities in vitro. We hypothesize that, if IGF-II is involved in similar activities during the wounding response, then the expression of IGF-II mRNA and protein, together with IGFBPs and IGF-Rs, will alter in the traumatized CNS. Furthermore, if disparate sites of IGF-II synthesis and bioactivity are maintained in the injured brain, then acutely, we would predict an increase in IGF-II secretion by the choroid plexus and in titer within the CSF, with the peptide being transported to sites of bioactivity in the wound. This transportation would be facilitated by the increased production of specific potentiating IGFBPs, both at the sites of synthesis and bioactivity. Completion of the cellular responses would be associated with (a) a decline in IGF-II bioactivity within wounds, (b) reduction in expression of ligands/receptors/potentiating binding proteins, and (c) an enhancement in expression of inhibitory binding proteins. Therefore, the aims of this study were to localize and quantify the levels of IGF-II and IGF-IIR mRNAs and peptides within CNS wounds and the choroid plexus by ribonuclease protection assay (RPA), in situ hybridization, immunohistochemistry and additionally, to quantify the levels of IGF-II and IGFBP-1–6 peptide in the CSF by RIA and Western blotting, during the cellular wounding response using a rat model of penetrating brain injury. We have previously reported on the changes in expression of IGF-1, IGF-IR, and IGFBPs within CNS wounds in the same model (10).
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Plasmids for complementary RNA (cRNA) probe synthesis
The 662-bp coding region of mouse IGF-II (a modified transcript
of one provided by V. Han, London, Ontario, Canada) is contained within
the pGEM-4Z plasmid (Promega Corp.). HindIII
and BglII were used to linearize the plasmid and SP6 and T7
were used to generate the transcript for antisense and sense templates
respectively. The 500 bp rat IGF-IIR fragment (D. LeRoith, Bethesda,
MD) is cloned within the pGEM-3 plasmid (Promega Corp.).
EcoRI and XbaI were used to linearize the
riboprobe, and T7 and SP6 generated the respective antisense and sense
templates. Cyclophilin (J. Douglass, Oregon Health Science University
of Medicine, Portland, OR) was an internal control in all ribonuclease
protection assays to ensure equal loading on gels. The plasmid contains
a 680 bp coding region of rat cyclophilin complementary DNA cloned
within pSP65 (Promega Corp.). Cyclophilin has a ubiquitous
tissue and phylogenetic distribution and represents 0.1–0.4% of total
cytosolic protein in many mammalian tissues. It possesses strong
homology to the enzyme peptidyl-prolyl cis-trans isomerase, which
catalyses the slow cis-trans isomerization of proline peptide bonds in
oligopeptides and accelerates slow, rate-limiting steps in the folding
of several proteins in the cell. HindIII was used to
linearize the plasmid for the antisense template. SP6 polymerase was
used to generate the antisense cRNA probe.
Antibodies and recombinant proteins
All antibodies used were IgG fractions of rabbit polyclonals,
except for IGFBP-5 (IgG fraction of an IGFBP-5 guinea pig polyclonal)
and IGF-II (mouse IgG monoclonal). Antibodies not raised against rat
proteins all have specified affinities for the corresponding rat ligand
(see supplier’s data sheet). Antihuman IGF-II antibody (Amano
Biologicals, Troy, CA) cross-reacted <1% with IGF-I and antirat
IGFBP-1 antibody (S. Shimasaki, San Diego, CA) had no known
cross-reaction with other binding proteins. Antibovine IGFBP-2 antibody
(Amano Biologicals) cross-reacted 0.1% with IGFBP-1, -3, -4, and -5,
whereas antirat IGFBP-3 antibody (S. Shimasaki) cross-reacted with no
other binding proteins, and antihuman IGFBP-4 antibody (TCS Biologicals
Ltd., Botolph Claydon, Buckinghamshire, UK) had a cross-reactivity with
IGFBP-1, -3, and -5 of 0.1–1% and up to 50% with IGFBP-2. Antihuman
IGFBP-5 antibody (TCS Biologicals Ltd.) had a cross-reactivity with
IGFBP-2 and IGFBP-3 of less than 1%, whereas antirat IGFBP-6 antibody
(S. Shimasaki) cross-reacted with no other known binding proteins.
Antirat IGF-IIR antibody (P. Nissley, Bethesda, MD) cross-reacted with
neither IGF-IR nor the insulin receptor. Recombinant human (h)IGF-I,
hIGFBP-1 and hIGFBP-3 for Western blotting were from TCS Biologicals
Ltd. Recombinant hIGFBP-2, -4, -5, and -6 were purchased from Amano
Biologicals. For RIA, recombinant hIGF-I and hIGF-II were purchased
from GroPep Ltd. (Adelaide, Australia). All antibodies were usually
stored at -20 C. If in frequent use, aliquots were stored at 4 C to
prevent repetitive freeze/thawing. All recombinant proteins were stored
at -70 C.
For immunohistochemistry. Antihuman IGF-II, IGFBP-4, and -5 antibodies were used at concentrations of 1:100, 1:3000, and 1:250 respectively, whereas antirat IGFBP-1, -3, -6, and IGF-IIR antibodies were used at 1:3500, 1:3000, 1:3500, and 1:2000, respectively. Antibovine IGFBP-2 antibody was used at the concentration of 1:3000.
For Western blotting. Antihuman IGFBP-4 and -5 antibodies were used at concentrations of 1:4000 and 1:1000, respectively, whereas antirat IGFBP-1, -3, and -6 were used at 1:4000. Additionally, antibovine IGFBP-2 and antirat IGF-IIR antibodies were used at the concentration of 1:4000 and 1:2000, respectively.
Animals and surgery
Surgery was performed aseptically under a British Government
Home Office Licence. Groups of adult, female, 250 g Sprague Dawley
rats were anesthetized ip with a mixture of Medetomidine
(SmithKline Beecham, Welwyn Garden City, Hertfordshire,
UK) at 100 µg/kg body weight and Ketamine hydrochloride
(Parke-Davis Veterinary, Eastleigh, Hampshire, UK) at 10
mg/kg body weight or Hypnorm (fentanyl nitrate 0.134 mg/liter and
fluanisone 10 mg/ml; Janssen Pharmaceuticals, Oxford,
UK)/Hypnovel (midazolam 1 ml in 10 ml water; Roche, Welwyn Garden City,
Hertfordshire, UK) at 8 ml/100 g body weight. Buprenorphine (Sterling
Health, Guildford, Surrey, UK) was administered postoperatively as an
analgesic. After craniotomy, the mediolateral right cerebral cortex was
incised using a David Kopf (Charles River, Margate, Kent, UK)
stereotactic instrument. The lesion was made precisely to a depth of 4
mm along a 4.5 mm line parallel with the sagittal suture, 3 mm lateral
to the mid-line, and spanning the fronto-parietal suture. Animals were
allowed to recover post surgery for periods of 0, 2, 5, 7, and 15 dpl
and fed ad libitum.
Sampling of CSF
Under deep anesthesia, an incision was made in the suboccipital
skin. The occipital semispinalis and trapezius muscles were separated
in the mid-line to expose the posterior atlanto-occipital membrane. A
Hamilton syringe needle was inserted through the membrane into the
cysterna magna and CSF withdrawn, samples from four animals were pooled
and frozen rapidly in liquid nitrogen and stored at -70 C until
processed.
Histology
Groups of four animals were deeply anesthetized (as for surgery)
and perfused transcardially with 250 ml of saline followed by 250 ml of
4% paraformaldehyde (PFA) in saline. Following excision, brains were
postfixed overnight in 4% PFA (wt/vol, in 0.1 M sodium
tetraborate) at 4 C, dehydrated in graded alcohols, embedded in low
melting point polyester wax (40), and stored at -70 C. Sections, 7
µm thick, were cut through the lesion site using a microtome (Bright,
Huntingdon, Cambridgeshire, UK) fitted with a cooled chuck, and mounted
on slides coated with either a 1% gelatin solution (for
immunohistochemistry), or with Biobond (British Biocell Int., Cardiff,
Glamorgan, UK; for in situ hybridization). Once mounted,
sections were air dried and stored at either -70 C (in situ
hybridization) or at 4 C (immunohistochemistry).
RNA extraction
Groups of three rats were killed with an anesthetic overdose,
the brains removed, rapidly dissected on ice, and stored at -70 C
until extraction. Total cellular RNA was extracted from the lesioned
and unlesioned hemispheres of the brain, using the RNAzol B method
(Biogenesis, Bournemouth, Dorset, UK), a modification of
the single-step method (41). Briefly, frozen tissue was weighed and
homogenized in 2 ml RNAzol per 100 mg tissue. Chloroform was added (0.2
ml/2 ml) and shaken for 15 sec before placing on ice for 5 min. Samples
were centrifuged at 5000 x g for 20 min at 4 C (Dupont
Sorvall RC5C, SS34 rotor, High Wycombe, Buckinghamshire, UK). The
aqueous phase was removed and precipitated with an equal volume of
isopropanol for 15 min on ice. Samples were centrifuged as above,
washed with 75% ethanol and redissolved in 200 µl ultrapure water.
RNA was quantified by optical density at 260 nm and 280 nm and checked
for integrity on a 1% agarose gel.
Radioactive probe synthesis
cRNA probes for ribonuclease assay. Transcription buffer
[40 mM Tris, pH 7.5, 6 mM MgCl2, 2
mM spermidine and 10 mM sodium chloride
(NaCl)], 20 U rRNasin, 10 mM dithiothreitol (DTT), 0.5
mM ATP, GTP, UTP, 12 µM CTP (150
µM for cyclophilin), 0.5 µg linearized antisense
plasmid, 50 µCi 32P CTP (5 µCi for cyclophilin) and 15
U RNA polymerase were incubated together at 37 C for 1 h. DNase I
(1 U) was added and the reaction left at 37 C for a further 15 min.
Yeast transfer (t)RNA (20 µg) was added before a
phenol:chloroform/chloroform extraction and ethanol precipitation. The
cRNA probe was washed with 75% ethanol, briefly dried, and resuspended
in ultrapure water. The probe, 1 µl in 5 ml Ecolite+ scintillation
fluid (ICN flow, High Wycombe, Buckinghamshire, UK), was counted using
a Pharmacia counter for 60 sec. 32P-labeled cRNA probes
were stored at -20 C for no longer than 1 week.
cRNA probes for in situ hybridization. Transcription buffer, 20 U rRNasin, 10 mM DTT, 2 mM ATP, GTP, CTP, 0.5 µg linearized antisense or sense plasmid, 200 µCi 35S UTP, and 15 U RNA polymerase were incubated together at 37 C for 2 h. DNase I (1 U) was added, and the reaction left at 37 C for a further 15 min. To the reaction mix, 60 mM EDTA pH 8.0 was added to a final volume of 50 µl, and loaded onto a Sephadex G50 Quick Spin column (Boehringer Mannheim, Lewes, E. Sussex, UK) which was centrifuged at 1100 x g (Hereaus Sepatech Varifuge 3.2RS, Brentwood, Essex, UK) for 4 min. DTT was added to a final concentration of 150 mM. The probe, 1 µl in 5 ml Ecolite+ scintillation fluid, was counted for 60 sec. 35S-labeled cRNA probes were stored at -20 C and kept for approximately 2 weeks. Before use, the 35S-labeled cRNA probes were recounted and their integrity checked on a 4% polyacrylamide/8 M urea gel.
125I labeling of IGF-I/IGF-II. Recombinant
hIGF-I and hIGF-II were labeled with 125I using the
chloramine-T method (42) under appropriate safety conditions. The
fractions were collected in 0.5 ml aliquots and 1 µl of each was
counted on a 
counter to locate the protein peak
(LKB-Pharmacia). The radiolabeled protein fractions were
stored at -20 C until required.
Ribonuclease protection assay
RPAs were performed on total RNA extracted from the cerebral
hemispheres of three rats from each treatment group. Total RNA (20
µg) was dissolved in 30 µl of hybridization solution (80%
formamide, 40 mM PIPES, pH 6.4, 400 mM NaCl and
1 mM EDTA, pH 8.0) containing 60,000 cpm and 20,000 cpm of
an IGF-related and cyclophilin 32P-labeled cRNA probe.
After being heated to 85 C for 5 min, the cRNA probe was allowed to
anneal the endogenous RNA at 45 C overnight. At the end of the
hybridization, the solution was diluted with 350 µl of RNase
digestion buffer (300 mM NaCl, 10 mM Tris, pH
7.4, and EDTA, pH 7.5), containing 40 µg/ml of RNase A and 500 U/ml
of RNase T1, and incubated for 1 h at 30 C. Proteinase K (100
µg) in 10% SDS was added to the sample and the mixture incubated at
37 C for an additional 20 min. Following a phenol:chloroform extraction
and ethanol precipitation, the pellet containing the RNA:RNA hybrid was
briefly dried and resuspended in loading buffer (80% formamide, 0.1%
xylene cyanol, 0.1% bromophenol blue and 2 mM EDTA, pH
8.0). The samples were boiled at 90 C for 5 min and separated on a 4%
polyacrylamide/8 M urea gel. 32P end-labeled (DNA
polymerase 1) HinfI digested pBR322 fragments were used as
molecular markers. The mRNA protected fragments were visualized by
autoradiography against Amersham Hyperfilm MP (Amersham International) at -70 C.
In situ hybridization
Mounted sections were dewaxed in ethanol, rehydrated, washed,
and digested with 10 µg/ml proteinase K in 0.1 M Tris
containing 50 mM EDTA at 37 C for 30 min. Sections were
rinsed in deionized water followed by an incubation in 0.1
M triethanolamine (TEA), pH 8.0, for 3 min. Sections were
then acetylated for 10 min with 0.25% acetic anhydride in 0.1
M TEA for 10 min, rinsed in 2 x SSC, dehydrated
through a graded series of ethanol washes, and air dried under vacuum
for 2 h before hybridization. Hybridization with the
35S-labeled cRNA probe (1 x 107 cpm/ml)
was performed at 55 C overnight in 10 mM Tris, pH 8.0,
containing 50% formamide, 0.3 M NaCl, 1 mM
EDTA, pH 8.0, 0.02% polyvinylpyrrolidone, 0.02% Ficoll, 0.02% BSA,
10 mM DTT, 0.05 mg/ml torula yeast RNA (C. P.
Laboratories, Bishops Stortford, Hertfordshire, UK), 0.5 µg/ml tRNA
and 10% dextran sulfate (wt/vol). After hybridization, sections were
rinsed for 1 h in 4 x SSC and treated with 25 µg/ml RNase
A in 10 mM Tris, pH 8.0, containing 0.5 M NaCl
and 1 mM EDTA pH 8.0 at 37 C for 30 min. This was followed
by increasing high stringency washes of SSC containing 1 mM
DTT, finishing with 0.1 x SSC at 65 C for 30 min. Slides were
then dehydrated through a graded series of ethanol, dried under vacuum,
and then exposed to Hyperfilm ß-max film for 10 days at 4 C to
examine gross changes in mRNA. For microscopic analysis, slides were
exposed to Ilford K5 liquid autoradiographic emulsion (Ilford Ltd.,
Basildon, Essex, UK) for 2 weeks at 4 C, processed with
Kodak D19 developer (Eastman Kodak,
Rochester, NY), rinsed and fixed with Kodak rapid fixer.
The slides were rinsed for 30 min in tap water, counterstained with
Mayer’s haemalum, examined by darkfield and brightfield microscopy
using a Zeiss Axioscope microscope [Carl Zeiss
(Oberkochen) Ltd., Welwyn Garden City, Hertfordshire, UK], and
photographed using Ilford PanF ISO 50 black and white film.
Immunohistochemistry
For immunoperoxidase staining (ABC Vectastain Elite
kit, Vector Laboratories, Inc., Peterborough,
Cambridgeshire, UK), tissue sections were dewaxed in 100% ethanol for
5 min and then rehydrated in 5-min steps in descending concentrations
of ethanol to ultra-pure water. Subsequent to a 5 min equilibration in
PBS, the endogenous peroxidase was quenched by incubating with 0.01%
hydrogen peroxide (H2O2) in PBS for 30 min. The
sections were rinsed in PBS and incubated in 1.5% goat serum (vol/vol,
Vector Laboratories, Inc.) diluted in PBS containing 0.1%
BSA for 30 min to block nonspecific staining.
After an overnight incubation at 4 C, with the appropriate concentration of growth factor, binding protein or receptor-protein-A-purified primary antibody diluted in PBS supplemented with 5% BSA, most sections were treated with a 1:200 dilution of biotinylated goat antirabbit IgG for 1 h, except for the IGFBP-5 antibody treated sections for which biotinylated antiguinea pig IgG and for IGF-II, for which biotinylated antimouse IgG were used. This was followed by 1 h incubation with the Vectastain Elite ABC reagent (Vector Laboratories, Inc.), a biotin-avidin-peroxidase complex. Finally, the sections were treated for 2–7 min with 0.5 mg/ml 3'3'-diaminobenzidine in PBS containing 0.01% H2O2. All steps were separated by PBS buffer washes. The sections were washed in PBS, counterstained with Mayer’s haemalum, dehydrated, and, after being cleared and mounted, examined by brightfield microscopy under differential interference contrast (DIC) optics, on a Zeiss Axioscope microscope and photographed using Fujicolor Super G plus ISO 200 color film (Fuji Photo Film Co., Ltd., Tokyo, Japan).
The specificity of the antibodies was verified by preincubating the primary antibody with excess (>1 µg) of the appropriate homologous or heterologous antigenic growth factor/binding protein. Sections were also processed with the primary or secondary antibody omitted. All of these controls yielded no visible staining of the sections. Antibodies to each binding protein were preincubated with every other recombinant binding protein species to determine specificity of staining.
Western blotting
CSF (2.5 µl or 10 µl from pooled samples from four animals)
was diluted with 97% sample buffer (0.16 M Tris HCl, pH
6.8, 22% glycerol, 6.1% SDS, 0.02% bromophenol blue, 0.02% xylene
cyanol). Rainbow colored (10 µl; Amersham International)
and unstained, low (and where appropriate high) molecular weight
molecular markers (37.5 µg; LMW electrophoresis calibration kit,
Pharmacia) and normal rat serum were diluted with 95%, 100% and 97%
sample buffer, respectively. Samples were heated to 100 C for 5 min and
immediately loaded onto a 0.1% SDS/12.5% polyacrylamide reducing gel.
The separated protein components were electroblotted in transfer buffer
(0.1 M Tris, 0.57 M glycine, 20% methanol
(vol/vol), pH 8.3) onto a Hybond C super (Amersham International) nitrocellulose membrane using an LKB
electroblotting unit (Pharmacia). A current of 0.8–1.0 A was applied
for 4 h at 4 C (or where appropriate overnight). The membranes
were air dried for 10–15 min. The marker lanes were removed from the
membrane and stained with amido black solution (0.1% amido black, 7%
acetic acid, 25% methanol) and then destained (10% acetic acid, 25%
methanol) until the background of the filter was white.
Membranes were incubated in Nonidet P-40 solution (5 mM Tris HCl, 0.05% Nonidet P-40, pH 7.4) for 30 min, BSA solution (5 mM Tris HCl, 3% BSA, pH 7.4) for 30 min, Tween 20 solution (5 mM Tris HCl, 15 mM NaCl, 0.2% Tween 20, pH 7.4) for 20 min, before being air dried. Radioactive (125I) IGF-I/IGF-II tracer at 10,000–16,000 cpm/100 µl was added to BSA solution and incubated with the membrane for 2 h. After discarding the radioactive probe, the membrane was washed in Tween 20 wash solution (50 mM Tris HCl, 0.15 M NaCl, 0.4% Tween 20, pH 7.4) for 15 min. This was followed by three washes of 15 min in Tris-buffered saline (50 mM Tris HCl, 0.15 M NaCl, pH 7.4). Membranes were air dried, covered in Saran wrap, and exposed to Kodak X-OMAT LS film at -80 C for 2–3 days. The film was processed in Kodak GBX developer, replenisher, and fixer.
The membranes were then blocked by shaking for at least 2 h in TBSTM (5% dried milk powder (Marvel, Premier Beverages, Stafford, Staffordshire, UK), dissolved in 15 mM Tris base, 0.22 M NaCl, 0.2% Tween 20, pH 7.4). During this time, the TBSTM was changed at least once. Membranes were placed in 10 ml/blot of primary antibody diluted in TBSTM, and rotated overnight. After two 30-min washes in TBSTM and one for 30 min in TBST, secondary antibodies were added. Antibodies were diluted to a final volume of 10 ml (antirabbit IgG-peroxidase 1:10000, antiguinea pig IgG-peroxidase 1:500) and placed with the membranes for 1 h. Membranes were washed three times for 15 min in TBST, followed by a brief rinse in ultra-pure water. An equal volume of Luminol (ECL detection kit; Amersham International) reagent 2 followed by Luminol reagent 1 was added. The solution was uniformly exposed to the membranes for 70 sec. All the Luminol reagents were removed, the membranes sealed in a plastic bag and placed immediately against Kodak X-OMAT LS film. The membranes were exposed against the film for approximately 1 h. Films were processed in Kodak GBX developer and replenisher.
RIA
IGF-II was measured in pooled samples of CSF from 4 rats by
specific RIA, as previously described (43) following removal of IGFBPs
by gel filtration chromatography on Sephadex G75 and elution with 1
M acetic acid. The following modifications were employed
from the published methods. The primary antiserum was a rabbit
antihuman IGF-II antibody (GroPep) and was used at a final
concentration of 1:1250. Recombinant hIGF-II (GroPep) was used as the
iodinated ligand for the standard curve (0.16 ng/ml to 20 ng/ml).
Incubation with the primary antiserum was for 3 days before centrifugal
separation of the bound and free tracer following the addition of
PEG-8000. The minimum level of detection of IGF-II was 0.6 ng/ml,
cross-reactivity with IGF-I was less than 1%, and the intra and
interassay coefficients of variation were 8% and 13%, respectively.
The recovery of IGF-II following gel chromatography was estimated as
greater than 90% as measured with the radiolabeled IGF-II added to
biological samples.
Densitometric and statistical analysis
Autoradiographic gels were scanned using a ScanJet IIc and
Deskscan II software (both from Hewlett-Packard Co.,
Geneva, Switzerland) into a Macintosh LC475 computer (Apple Computer
Inc., Cupertino, CA). On RPA autoradiographs, bands corresponding to
the protected mRNA fragments of IGFs, IGFBPs, IGF-Rs and cyclophilin,
were densitometrically quantified into arbitrary units using NIH Image
analysis software (NIH). The IGF-related bands of interest were
normalized by dividing IGF-related mRNA pixel values by cyclophilin
mRNA values. Likewise, similar quantifications were performed on
Western blot autoradiographs, on bands indicating IGF-ligand or
IGFBP-antibody binding. Where appropriate, both means and
SEM were calculated and plotted. Significant differences in
relation to data from either the 0 dpl control, or the contralateral
unlesioned hemisphere, were examined using single-factor ANOVA with a
significance (
) level of 5%.
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and 2,
respectively. All data mentioned, but not present herein, are available
for inspection on written request to the corresponding author. The
localization of IGF-I, IGFBP-1–6, and IGF-IR mRNA and peptide, in
unlesioned and lesioned adult rat brains, has been catalogued by us in
detail elsewhere (10), as has the localization of IGF-II throughout the
intact brain (4). IGF-II and IGF-IIR mRNA and peptide expression
altered in response to injury, as did corresponding levels of IGF-II
peptide within the CSF. IGFBP-2, -3, and -6 peptides were also observed
within the CSF. Here, levels of IGFBP-2 predominated and were injury
responsive. Because the corresponding IGF-II and IGFBP mRNAs were
expressed in the choroid plexus, this site was a likely source of these
proteins. No IGF-I, IGFBP-1, IGFBP-4, or IGFBP-5 peptide were detected
in the CSF by the methods described here.
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and 2).
In the acute phase (1–7 dpl), IGF-II mRNA levels did not increase
within any cells of the damaged neuropil, whereas in the chronic phase
mRNA expression was strong and specifically localized within the
marginal astrocytes of the forming glia limitans of the scar (Figs. 1C and 2, G–H).
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, A and B). Additionally, after 7 dpl,
most of the astrocytes of the forming glia limitans became strongly
IGF-II immunoreactive (Fig. 3C). Additionally, IGF-II peptide was also
detected within CSF samples by RIA. Low levels of IGF-II were present
within CSF samples from unlesioned brains, with the levels
increasing transiently post injury to peak at 7 dpl (Fig. 4).
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). A predominant probable IGFBP-2 band
was observed at approximately 30 kDa, with two lesser bands between
39–43 kDa, which may have been IGFBP-3, and a faint band at 20 kDa
(possibly IGFBP-6) that was unquantifiable (n = 2).
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compared with the 0 d control (Fig. 6, A and B). The response was transient,
peaking at 7–10 dpl and declining thereafter. A small biphasic
increase in ligand binding was also observed in the CSF in the 39–43
kDa bands with peaks at 2 and 15 dpl compared with the 0 d. (Fig. 5B
).
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) in the CSF
from intact as well as lesioned brains. The 32-kDa band followed a
similar pattern of increase to that of the 30 kDa protein on the ligand
blot, suggesting that this was intact IGFBP-2. The levels of this BP
increased from 2 dpl to peak between 7–10 dpl and thereafter declined
(Fig. 7B). The smaller 18 kDa peptide, which did not bind ligand and
probably represents a residual fragment of proteolytically cleaved
IGFBP-2, showed a sustained increase in expression during the full
time course of the experiment (Fig. 7B).
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IGFBP-6
Using IGFBP-6 antibodies, one band was visible at approximately 23
kDa, probably corresponding to the 20-kDa protein band observed on the
ligand blot. Titers of immunoreactive IGFBP-6 protein showed a small
increase at 2 dpl, but values subsequently declined and by 10 dpl
levels equivalent to those observed in the 0 d control sample were
recorded (data not shown).
IGF-II receptor in the cerebral hemispheres
A 500-bp protected mRNA species corresponding to IGF-IIR was
identified by RPA in all samples. There were no significant changes in
mRNA expression in any of the unlesioned or lesioned hemispheres when
examined by RPA (data not shown). By in situ hybridization,
low levels of IGF-IIR mRNA were detected diffusely throughout the brain
(Fig. 8), barely above background, with
high levels of expression localized to the piriform cortex, internal
capsule, meninges and ventricular choroid plexi, in both the intact and
lesioned brain (Fig. 9, A–D). Within the
wound, changes in IGF-IIR mRNA in response to injury were identified,
but the response was strictly localized to the astrocytes of the
maturing glia limitans and only became apparent at 12 dpl (Fig. 9, G–H).
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, E and F),
although immunopositive IGF-IIR staining appeared at 12 dpl to be
associated with the astrocytes comprising the maturing glia limitans
(Fig. 3G). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In our previous description of the normal distribution of IGF-II in the adult rat brain (4), IGF-II mRNA was seen to be expressed in the highly vascularized areas of the mesenchymal support structures, predominantly in the meninges, microvasculature, and the ventricular choroid plexi. Additional levels of high expression were also observed within the hippocampus and thalamus. Colocalized IGF-II peptide in areas of high mRNA expression, indicated mRNA translation (4). Thus, IGF-II expression was, in part, associated with structures that are involved in the production of extracellular fluids (including CSF), which are responsible for substrate transport and supply in the CNS (4, 5). The presence of IGF-II protein in CSF has previously been demonstrated, together with a number of putative transport proteins (44). The coexpression of IGFBP-2 by cells of the choroid plexus and meninges and the corresponding detection of significant levels of IGFBP-2 peptide coincident with IGF-II within the CSF (4, 18, 45, 46) adds weight to the hypothesis that IGFBP-2 is a key transport protein for IGF-II in the CNS, mediating its transfer via the CSF from sites of synthesis to sites of storage and/or bioactivity. Certainly, this hypothesis would explain the localization of immunoreactive IGF-II and IGFBP-2 in the absence of significant levels of their mRNA at sites throughout the brain (4). Furthermore, this pattern of differential siting of IGF-II expression and bioactivity is reminiscent of the endocrine delivery of hormones.
This study demonstrates that, despite substantial changes in the IGF-II axis after a penetrating injury to the adult rat brain, the endocrine-like mode of delivery is maintained. Hence, after injury, the bioavailability of IGF-II in wounds is altered, not by local changes in its mRNA expression, but through changes of IGFBP expression and the ensuing import of the IGF-II peptide. Here, we discuss a putative endocrine action of IGF-II in CNS wounds, which differs from the autocrine/paracrine mechanisms previously discussed by us for IGF-I (10).
The acute response of IGF-II, IGFBPs, and IGF-Rs to penetrating CNS
injury
In the acute phase response to a penetrating CNS injury (1–7
dpl), IGF-II appears to support the autocrine/paracrine actions of
IGF-I in the wound, behaving as a neurotrophin and gliotrophin. The
rapid mobilization of IGF-II peptide into the damaged neural parenchyma
in the absence of increased local mRNA expression, occurs when cellular
activity within the wound is maximal. Therefore, transport for IGF-II,
from its site of synthesis in the mesenchymal support structures of the
brain to its distal site of action, must occur.
IGF-II, IGFBP-2, IGFBP-3, and IGFBP-6 mRNA are all expressed by the choroid plexi throughout the ventricular system, a likely source of the peptides found within the CSF. The presence of IGF-I peptide was not detected in the CSF, by RIA, in any of the samples taken from unlesioned or lesioned adult rat brains (data not shown; positive control detectable at 0.4 ng/ml). We have detected IGF-II, IGFBP-2, IGFBP-3, and IGFBP-6 peptides within the CSF, with increased levels of each occurring during the acute phase response to injury. However, within the CSF, only IGFBP-2 binds IGF ligands with any significance after injury. With the disruption of the blood-brain barrier that occurs in penetrating wounds, it remains uncertain whether the slightly increased levels of IGFBP-3 and IGFBP-6 are serum derived. From the observed changes in IGFBP titers in the CSF, IGFBP-2 would seem to be the major mediator of IGF-II transport. Within the local parenchyma of the lesion, we have shown that all IGFBPs are present (10) and, therefore, postulate that they are involved in local sequestration and/or modulation of IGF activity within wounds. Interestingly, we have previously shown increased levels of IGFBP-2 mRNA in the wound parenchyma (10), suggesting that additional expression by injury responsive cells may locally regulate IGF actions at this site.
In summary, these studies suggest that there is an acute phase increase in production of IGF-II away from the site of injury by the choroid plexus cells, from where it is transported via the CSF to localize within a CNS wound. Within the CSF, IGF-II is predominately bound by IGFBP-2 and is biologically inert, but changes in the local equilibrium of IGFBPs at the lesion site make it physiologically available to target cells possessing IGF-IR and IGF-IIR including glia and neurons (10). It is possible that IGF-II may also be mobilized both from the myelin sheaths of isolated axons and from myelinated nerve tracts, where the peptide is normally sequestered. Whatever mechanism is implemented, between 3–7 dpl, IGF-II has the potential to function in CNS wounds as a key acute phase endocrine regulator of astrogliosis and neuronal sprouting.
Chronic actions of IGF-II, IGFBPs and IGF-Rs in response to a
penetrating CNS injury
During the chronic response to a CNS injury, occurring between
7–15 dpl, IGF-II levels also decline within the CSF, thereby
decreasing the supply of bioavailable IGF-II to the wound.
Additionally, within the CSF, IGFBP-2 decreases, although the level of
the IGFBP-2 18 kDa fragment increases. This fragment, which does not
bind IGF-II and may be a product of IGFBP-2 proteolysis within the CSF,
may signify attenuation of IGF-II bioactivity.
Within the damaged neural parenchyma of the cerebral wound, the levels of IGF-II and IGFBP-2, -3, and -6 peptides decrease as the wound matures (see also 10). However, at 7 dpl, we observed increasing levels of IGF-II mRNA, colocalized with IGF-II protein, to a specific population of astrocytes bordering the wound. These astrocytes show phenotypic changes as they migrate and secrete matrix components initializing the formation of a glia limitans within the wound, eventually delineating the wound margins to becomes contiguous with the external glia limitans of the brain. The constitutive expression of IGF-II becomes a feature of this selected population of astrocytes, as it is for the astrocytes comprising the glia limitans externa. At this site and elsewhere in the intact and lesioned brain we have shown that the constitutive expression of IGF-II and IGFBP-2 are spatially and temporally coincident (4).
The levels of IGF-IR mRNA expression remain unaltered in response to a penetrating injury to the CNS (10), suggesting that changes in IGF-IR expression are not a primary determinant of IGF bioactivity. We presume that increased IGF-IIR levels in injury responsive astrocytes and neurons in the acute wound (2–5 dpl) derive from distant sites of synthesis because no corresponding mRNA was detectable. Although a soluble form of the IGF-IIR peptide has been identified in a variety of serum sources (47, 48), we were unable to detect its presence within the CSF, by Western blotting, in samples taken from unlesioned and lesioned adult rat brains (data not shown). However, during the chronic response to injury (after 12 dpl), IGF-IIR mRNA expression did increase focally with IGF-II mRNA within selected astrocytes comprising the maturing glia limitans of the scar in a similar manner to that of IGF-II and IGFBP-2. Interestingly, increased expression of IGFBP-5 mRNA has also been demonstrated in the reforming glia limitans of the injured brain at the same time (10). IGFBP-5 reportedly inhibits IGF in many tissue systems (9) and may assist in the sequestration of IGF-II in the wound during the later phase of the injury response, thereby modulating IGF-II activity at this site. The highly coordinated expression of these interacting proteins in the chronic CNS lesion implicates a role for these factors in the reestablishment and maintenance of the integrity of the glia limitans. Therefore, in the chronic phase of the CNS wounding response, IGF-II may reassert its actions as an autocrine/paracrine factor within the glia limitans of the wound, in contrast to the acute phase endocrine action seen locally in the neural parenchyma.
In summary, a second mechanism for IGF-II action in the chronic phase of CNS injury is proposed. In the intact brain, constitutive IGF-II and IGFBP-2 mRNA and protein expression are a phenotypic feature of the astrocytes present within the glia limitans externa. It is possible that their role is to maintain homeostasis in the mesenchymal support structures of the brain, primarily by an autocrine/paracrine mechanism. In the chronic phase response to injury, after the endocrine-like acute phase response in the wound neuropil, we postulate that IGF-II reverts to its primary role as an autocrine/paracrine factor in the maturing glial membranes of the wound. Hence, at this time, levels of IGF-II mRNA increase to constitutive levels focally in the differentiating astrocytes of the wound glia limitans thereby helping to restore tissue homeostasis.
In conclusion, we propose that within the uninjured brain IGF-II expression in the mesenchymal support structures is associated with an autocrine/paracrine role in tissue homeostasis. IGF-II and IGFBP-2 mRNA are constitutively expressed by the choroid plexus throughout the brain. The presence of immunoreactive IGF-II within the CSF and localization within myelinated tracts under normal physiology suggests circulation within the former and sequestration at the latter distal depots; a pattern of transport and deposition which is maintained after injury. After a penetrating CNS injury, this pattern of expression is maintained with IGF-II synthesized at sites distal to its sites of bioactivity. During the acute phase of the wounding response, IGF-II expression is up-regulated in the choroid plexus cells, which leads to the increased secretion of the peptide into the CSF. Consequentially, there is an increase in transportation to the wound of IGF-II complexed to IGFBP-2, where it may target receptor-bearing glia and neurons. Hence, an essentially endocrine mode of action is indicated for IGF-II during the early phases of CNS wounding, with a balance of locally produced stimulatory/inhibitory IGFBPs modulating its bioactivity. Later in the response, as the IGF-II levels drop in the neural parenchyma around the wound, IGF-II may reassert its autocrine/paracrine role in the maintenance of tissue homeostasis via actions at the glial membrane.
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Acknowledgments |
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Footnotes |
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Received June 8, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, 32 kDa,
IGFBP-2 protein band.
