Single-day therapy: an expert opinion on a recent development for the episodic treatment of recurrent genital herpes

One common method for treating recurrent genital herpes outbreaks is 3–5 day episodic therapy with nucleoside analogues. However, since maximum viral replication occurs within 24 h after the onset of symptoms, short-term patient-initiated episodic therapy started at prodromal onset or at the first appearance of lesions in patients without a prodrome may represent an important option. In a recent randomized trial, single-day famciclovir treatment decreased lesion healing time and the duration of pain and other symptoms by approximately 2 days compared to placebo, and prevented progression to a full outbreak in almost one in four patients. Because single-day treatment is more convenient than traditional therapies, it may lead to improved patient compliance and better overall management of recurrent genital herpes outbreaks.
Introduction
Recurrent genital herpes outbreaks are predominantly caused by herpes simplex virus type 2 (HSV-2), although the incidence of genital herpes caused by herpes simplex virus type 1 (HSV-1) is rising due to changing attitudes about oral-genital sexual behavior [10]. Approximately one in four adults in the United States is seropositive for HSV-2 [7], an incurable infection that initially enters the body through mucous membranes or abraded skin. Once primary infection has occurred, the virus remains dormant in the dorsal root ganglia, where it can reactivate to cause uncomfortable, recurrent outbreaks [20].
HSV-2 infection is somewhat more prevalent in women than in men [7], and can have a profound psychological impact as well as physical repercussions, resulting in a decrease in measures of quality of life. The fear of spreading the infection can result in lowered self-esteem or sexual desire and, less commonly, social phobia and depression [6]. In addition, patients with genital herpes have a significantly increased risk of acquiring human immunodeficiency virus (HIV) [8].
Episodic treatment for genital herpes
Although there is no cure for HSV-2 infection, patients have two treatment options: treating outbreaks as they occur (episodic therapy), or attempting to prevent future outbreaks (suppressive therapy). Suppression with daily oral antivirals may be most appropriate for patients with frequent or uncomfortable recurrences and to reduce genital herpes transmission to sexual partners [4]. Although suppressive therapy is effective, one quarter to one half of patients on suppressive therapy have at least one recurrence per year [5, 14].
Although episodic treatment has not been proven to reduce the risk of transmission, it still remains an important option for patients who do not want to take daily suppressive therapy, are not concerned about the frequency of recurrences, or are not sexually active. The best method for administering episodic therapy is to make medication readily available to patients to self-administer at the first onset of symptoms or lesions [16]. This type of patient-initiated episodic therapy has been successfully used in previous clinical trials of recurrent genital herpes [13, 15]. The ability to treat recurrences sooner may also help decrease the duration of an outbreak or halt progression to a full outbreak [9, 13, 15, 19]. This expert opinion will examine the results from a recently published clinical trial about patient-initiated, single-day oral antiviral therapy (famciclovir) for recurrent genital herpes [2] and compare its efficacy with data from currently available therapies.
Treatment of recurrent genital herpes with oral antivirals
Patients with recurrent episodes of genital herpes often experience such prodromal symptoms as itching, tingling, burning, and pain, with papules and vesicles typically forming 12–24 h after onset of these symptoms [3]. Maximum HSV-2 concentration and lesion pain usually occur within the first 24 h of onset of these prodromal symptoms [3]. Under the assumption that one can extrapolate from the pathogenesis of herpes labialis [17], it is thought that the most effective way to treat recurrent genital herpes with antiviral therapy would be within the first 24 h after the onset of clinical symptoms, when viral replication is highest [16]. Oral antivirals, acting as nucleoside analogues enter infected cells and bind to viral thymidine kinase, resulting in their phosphorylation. After the antivirals are further phosphorylated by cellular enzymes, they compete with nucleosides to bind to the viral DNA polymerase, resulting in polymerase inactivation and a decrease in viral replication [16].
Although topical acyclovir was initially used to treat recurrent genital herpes, it has since been proven ineffective and is no longer recommended [1, 12]. Today, the most commonly prescribed drugs are the oral antivirals acyclovir, valacyclovir, and famciclovir. Acyclovir is approved for 5-day episodic treatment, although a 2-day regimen has been shown to be effective [13, 19]. Acyclovir is effective, but its poor bioavailability requires more frequent dosing. Valacyclovir, a prodrug of acyclovir, has a higher bioavailability than acyclovir and has been approved for 3-day episodic treatment [9, 14]. Famciclovir, a prodrug of penciclovir, is currently indicated for 5-day treatment [11, 15]. The high bioavailability of famciclovir (77%) and the rapid onset of viral replication in recurrent genital herpes suggested that this medication would be efficacious in an even shorter single-day treatment regimen.
Data from single-day famciclovir clinical trial
A clinical trial was performed to assess whether single-day famciclovir was effective in treating recurrent genital herpes in immunocompetent individuals [2]. The 329 study participants experiencing a recurrence of genital herpes were predominately female (71%) and 48% had had more than six recurrences in the preceding year. Participants were given a single day of famciclovir 1,000 mg bid or matching placebo and were instructed to take the medication within 6 h of the onset of prodromal symptoms and/or genital herpes lesions during their next recurrence. The trial results showed that a single-day regimen of famciclovir significantly decreased lesion-healing time and significantly reduced time to resolution of all symptoms by 2 days, as compared to placebo. Famciclovir also halted the progression to a full genital herpes outbreak in almost one in four patients. Adverse events were of mild-to-moderate intensity and were similar to the adverse events experienced by the placebo group.
Although no head-to-head studies have been conducted between single-day famciclovir and other oral antivirals, patients who received single-day famciclovir experienced effects similar to those seen in previous clinical trials of traditional longer-term therapies (Table 1) [2, 9, 13, 15, 18, 19]. Single-day therapy appears to inhibit viral replication enough to significantly reduce both symptoms and the tissue damage characteristic of a full outbreak, preventing progression to a full recurrence in some cases.
Table 1Effectiveness of oral antivirals in clinical trials of episodic treatment for genital herpesDrugTreatment regimenMedian time (days) to lesion healing (treatment vs control)Percent of aborted episodes (treatment vs control)Acyclovir [13, 19]800 mg tid × 2 days versus placebo200 mg 5 times daily × 5 days versus placebo4.0 versus 6.0 (P = .001) 5.7 versus 7.2a (P ≤ .001)27.0 versus 10.6% (P = .029)    Was not performedValacyclovir [9, 18]500 mg bid × 3 days versus 500 mg bid × 5 days1,000 mg bid × 5 days versus 200 mg acyclovir 5 times daily × 5 days versus placebo4.4 versus 4.7 (P = NS)4.8 versus 4.8 versus 6.0 (P < .001)b25.4 versus 26.6% (P = NS)25.9 versus 24.8 versus 19.8% (P = NS)Famciclovir [2, 15]1,000 mg bid × 1 day versus placebo125 mg bid × 5 days versus placebo4.3 versus 6.1 (P < .001)3.8 versus 4.8 (P < .001)23.3 versus 12.7% (P = .003)Was not performedaMean time to healingbP values correspond to both valacyclovir versus placebo and acyclovir versus placeboNS = Not significant
Conclusion
The rapid onset of viral replication in recurrent genital herpes suggests that a shorter course of therapy would be as efficacious as traditional treatments. Patient-initiated single-day therapy could prove helpful for obstetricians/gynecologists who treat patients with genital herpes, as it may provide a more convenient option for treating the disease, and could help to promote patient compliance. High bioavailability, ease of use, and the added benefit of preventing progression to a full genital herpes outbreak in some patients make famciclovir an excellent candidate for single-day patient-initiated episodic therapy.

genital herpes
patient-initiated episodic therapy
single-day famciclovir


Mast cell-specific Cre/loxP-mediated recombination in vivo

Mast cells are important effectors of type I allergy but also essential regulators of innate and adaptive immune responses. The aim of this study was to develop a Cre recombinase-expressing mouse line that allows mast cell-specific inactivation of genes in vivo. Following a BAC transgenic approach, Cre was expressed under the control of the mast cell protease (Mcpt) 5 promoter. Mcpt5-Cre transgenic mice were crossed to the ROSA26-EYFP Cre excision reporter strain. Efficient Cre-mediated recombination was observed in mast cells from the peritoneal cavity and the skin while only minimal reporter gene expression was detected outside the mast cell compartment. Our results show that the Mcpt5 promoter can drive Cre expression in a mast cell-specific fashion. We expect that our Mcpt5-Cre mice will be a useful tool for the investigation of mast cell biology.
Introduction
Mast cells are central effector cells in type I allergy (Kawakami and Galli 2002; Metcalfe et al. 1997), but have recently been shown to be important initiators and effectors also of innate immunity as well as modulators of adaptive immune responses (Galli et al. 2005a, b; Marshall and Jawdat 2004). Many reports demonstrated a role of mast cells in host defence against pathogens (Dawicki and Marshall 2007; Echtenacher et al. 1996; Malaviya et al. 1996) and implicated a function of these cells in wound healing, tissue remodeling and transplant tolerance (Lu et al. 2006; Trautmann et al. 2000; Weller et al. 2006). A pathogenic role of mast cells was discussed in rheumatoid arthritis, systemic sclerosis, multiple sclerosis and atherosclerosis (Bachelet et al. 2006; Irani et al. 1992; Lee et al. 2002; Secor et al. 2000; Sun et al. 2007).
Investigation of mast cell biology was severely hampered by the scarcity of these cells in tissues. Experimental systems that have widely been used are in vitro analysis of mast cell lines or mast cells differentiated in vitro from bone marrow (BMMC). In vivo analysis of the function of genes in mast cells until today relied on the reconstitution of mast cell-deficient rodents with mast cells derived from bone marrow or hematopoietic cells of gene-deficient animals or from gene-deficient embryonic stem cells (Kitamura et al. 1978; Nakano et al. 1985; Tsai et al. 2002, 2000). These systems yielded valuable information, but suffer important limitations. In the mouse lines kitW/W-v and kitW-sh/W-sh, which are commonly used for reconstitution experiments, spontaneous mutations of the kit gene coding region or its regulatory elements result in reduced activity of the kit receptor that controls development and survival of mast cells (Galli et al. 2005b; Grimbaldeston et al. 2005). In addition to mast cell-deficiency, kitW/W-vanimals are characterized by sterility, anemia, absence of melanocytes and interstitial cells of Cajal, a high incidence of dermatitis, papillomas of the forestomach, gastric ulcers and dilation of the duodenum (Galli et al. 2005b). kitW-sh animals, which lately are being used instead of kitW/W-v mice, seem to feature a more narrow defect with deficiency for mast cells, melanocytes, and interstitial cells of Cajal (Grimbaldeston et al. 2005). The transfer of in vitro cultivated mast cells into mast cell-deficient animals by intradermal, intraperitoneal or intravenous injections can result in mast cell numbers, which, dependent on the route of cell transfer and the anatomical site, can approach normal mast cell density, but often remain considerably lower with significant variability (Grimbaldeston et al. 2005; Tsai et al. 2005).
The Cre/loxP recombination system represents a powerful tool that allows for conditional, e.g. cell type-specific, inactivation of genes in the mouse (Rajewsky et al. 1996). Since the first description of in vivo mutagenesis using the Cre/loxP system (Gu et al. 1994), an impressive number of floxed mouse lines has been generated. Herein, we report the first Cre transgenic line allowing mast cell-specific gene inactivation in vivo.
Material and methods
Construction of the transgene and generation of transgenic mice
The construct was based on a bacterial artificial chromosome (BAC) encompassing the entire Mcpt5 gene. The BAC clone RP23-284A14 from the mouse BAC library RPCI-23 (Osoegawa et al. 2000) was obtained from RZPD (Deutsches Ressourcenzentrum für Genomforschung GmbH, Berlin, Germany). All modifications were done by homologous recombination in E. coli (Zhang et al. 1998) and sequences of all oligonucleotides used for construction by homologous recombination are listed in Table 1. Neighbouring genes contained in the BAC were excluded by replacing several kb of DNA at both ends of the insert by resistance genes (Fig. 1). In a next step, a modified iCre (improved Cre, Shimshek et al. 2002) gene cassette (Testa et al. unpublished) was inserted into the Mcpt5 gene. The Cre cassette was first assembled with a loxm2-flanked neomycin-resistance cassette (neo) and flanked by homology regions (HR) for recombination into the Mcpt5 gene. This was done by subcloning the Cre cDNA into a minimal vector consisting of the origin of replication and a chloramphenicol resistance gene amplified from pACYC184 (New England Biolabs, Frankfurt, Germany). The primers used for the amplification contain the HRs for both subcloning of Cre and insertion into the Mcpt5 gene (see Table 1). In a next step, the neomycin cassette was inserted 3′ of Cre and 5′ of the Mcpt5 HR. The HR-Cre-Neo-HR cassette was released from the minimal vector by NotI digestion and recombined into the BAC replacing the coding part of exon 1 of the Mcpt5 gene followed by Cre-mediated deletion of neo. The construct was purified for pronucleus injection as described by Sparwasser et al. (2004) with modifications. Briefly, the construct was separated from the BAC backbone by NotI digest and purified by gel electrophoresis and electroelution. Pronucleus injection was done by R. Naumann, MPI of Molecular Cell Biology and Genetics, Dresden, Germany. Genotyping of Mcpt5-Cre transgenic mice was performed by PCR (primers: Mcpt5-CreFor 5′ACAGTGGTATTCCCGGGGAGTGT, Mcpt5-CreRev 5′ GTCAGTGCGTTCAAAGGCCA). Founder mice were bred to the ROSA26-EYFP reporter line (Srinivas et al. 2001) and five to 15 week old mice were analyzed for reporter gene expression. Animals were housed under SPF conditions and all experiments were done according to institutional guidelines.
Table 1Primers used for transgene construction by homologous recombinationOligonucleotides used for transgene construction by homologous recombination. The respective 50 bp homology regions (HR) were included into the primers used for the amplification of selection markers or the minimal vectorFig. 1Strategy of transgene construction. The BAC RP23-284A14 was shortened by replacing 51.3 kb of insert DNA at the T7 end of the vector backbone and 42.4 kb at the SP6 end by an ampicillin (Amp) and zeocin (Zeo) resistance cassette, respectively, resulting in a residual insert length of 129 kb. A NotI restriction site was inserted along with each of the resistance genes. The coding part of the first exon of Mcpt5 was replaced by a cassette comprising the Cre cassette and a loxm2-flanked neomycin resistance cassette (Neo) by homologous recombination and subsequent deletion of neo by Cre-mediated deletion in E. coli. (NotI, N, EcoRI, I, arrows represent open reading frames, shaded boxes represent untranslated regions)
Flow cytometric analysis
Total splenocyte suspensions were separated into lymphocyte and non-lymphocyte fractions by magnetic cell sorting (MACS, Miltenyi Biotec, Bergisch Gladbach, Germany) using anti-CD3-Biotin (clone 145-2C11, BD Pharmingen), anti-Biotin MicroBeads, anti-CD19 MicroBeads and LS MACS columns (Miltenyi Biotec). Skin cell suspensions were prepared from shaved abdominal skin plus one ear pinna by mincing followed by an incubation with 2 mg/ml collagenase IV (Worthington Biochem. Corporation, Lakewood, NJ, USA) at 37°C. Cells in peritoneal lavage as well as single cell suspensions from skin and spleen were stained with phycoerythrin (PE)-conjugated anti-CD3 (clone 145-2C11), anti-CD19 (clone 1D3); anti-CD11c (clone HL3), anti-CD45 (clone 30-F11) and anti-Siglec-F (clone E50-2440) (all from BD Pharmingen) or anti-FcεRIα-PE (clone MAR-1), allophycocyanin (APC)-conjugated anti-CD117 (clone 2B8) and anti-CD49b (clone D×5), F4/80-Biotin (clone BM8) and streptavidin-PE (all from eBiosciences, SanDiego, CA) or anti-Gr-1-PE (clone RB6-8C5) (Miltenyi Biotech). The cells were analyzed on a FACSCalibur (BD Biosciences).
Results
Transgene construction and screening of transgenic mice for Cre expression
In order to drive Cre expression in mast cells, we used a BAC clone containing the entire Mcpt5 gene along with abundant upstream and downstream flanking DNA (Fig. 1). Neighboring genes were excluded by shortening the BAC on both ends to avoid possible effects of altered gene dosage in the transgenic mice. In a next step, the coding part of the first exon of Mcpt5 was replaced by the Cre cassette (see Methods and Fig. 1). The construct was released from the BAC backbone by NotI digest (Fig. 1) and injected into the pronuclei of C57BL/6 oocytes. Six Mcpt5-Cre transgenic founder animals were obtained and mated to a ROSA26 reporter line (Srinivas et al. 2001) which expresses enhanced yellow fluorescent protein (EYFP) under the control of the ubiquitous ROSA26 promoter following Cre-mediated deletion of a loxP-flanked stop element. Mcpt5-CreROSA26-EYFP double transgenic animals were analyzed for EYFP expression in peritoneal and skin mast cells by FACS as described below (Fig. 2). Reporter gene expression was detected in mast cells from three of the six founder lines. One of these lines showed EYFP expression in only 80% of peritoneal mast cells and was not investigated further. Close to 100% of peritoneal mast cells were EYFP positive in two lines, which, because of autosomal inheritance of the transgene in one line and X-linked inheritance in the other, were called “A-Mcpt5-Cre” and “X-Mcpt5-Cre”, respectively.
Fig. 2Efficiency of Cre-mediated reporter gene activation in mast cells. EYFP expression is demonstrated in cells from peritoneal lavage fluid and single cell suspensions of skin of Mcpt5-Cre ROSA26-EYFP double transgenic mice. Peritoneal mast cells were stained for CD117 and FcεRIα (n = 7) and skin mast cells for CD117 and CD45 (n = 4). Mast cell populations were gated and their EYFP fluorescence displayed in histogram plots. The black graph represents Mcpt5-Cre ROSA26-EYFP double transgenic mice, the shaded graph represents the Cre-negative but ROSA26-EYFP-positive control (littermates in most instances)
Efficient and mast cell-specific Cre-mediated recombination in the lines A-Mcpt5-Cre and X-Mcpt5-Cre
Highly efficient Cre-mediated activation of reporter gene expression was uniformly observed in peritoneal CD117+FcεRIα+ mast cells of seven A-Mcpt5-Cre ROSA26-EYFP double transgenic animals (98.7–99.6%, on average 99.1%, Fig. 2). In skin cell suspensions mast cells were detected in extremely low numbers, on average 0.32%. (Skin mast cells were defined as CD117+CD45+ cells. A staining of FcεRIα on skin mast cells was not reliable in our hands, likely due to loss of epitopes after enzymatic tissue digestion). In the five animals for which this mast cell population was investigated, the majority of the few CD117+CD45+ cells gated was EYFP positive (43/47, 54/64, 32/39, 23/27 and 77/88 gated events, Fig. 2). Given that the mast cell gate will inevitably collect also some non-mast cells upon analysis of high total cell numbers (40,000 events), the percentage of skin mast cells expressing EYFP is probably close to 100%. A similar efficiency of Cre-mediated activation of the reporter gene was observed in three male X-Mcpt5-Cre mice (not shown) whereas the two female animals analyzed showed EYFP fluorescence in only 61% and 32% of peritoneal mast cells consistent with random X-chromosome inactivation (not shown).
In both lines, Cre-mediated activation of EYFP expression was specific for mast cells. In seven A-Mcpt5-Cre and three male X-Mcpt5-Cre animals we detected no or only minimal numbers (i.e. below 1%) of EYFP positive cells (below 3% in one A-Mcpt5-Cre animal) in hematopoietic populations other than mast cells (Fig. 3) or in non-hematopoietic cells of the skin cell suspensions (primarily keratinocytes and fibroblasts, not shown). As a control, we used mice with germline deletion of the stop element of the ROSA26-EYFP Cre excision reporter demonstrating that all non-mast cell populations analysed for reporter expression in Mcpt5-Cre mice can, in principle, express EYFP (not shown). In contrast to the seven A-Mcpt5-Cre mice (described above) showing mast cell-specific Cre-mediated recombination, one A-Mcpt5-Cre mouse displayed non-specific reporter gene expression in 15–25% of all cell types analyzed including non-hematopoietic cells of the skin (not shown). This finding likely reflects accidental early activity of the transgenic Mcpt5 promoter.
Fig. 3Absence of EYFP reporter gene expression in hematopoietic cells other than mast cells. EYFP expression was analyzed in hematopoietic cells from peritoneal lavage and spleen cell suspensions of Mcpt5-Cre ROSA26-EYFP double transgenic mice. In order to enrich granulocytes, splenocytes were separated into a lymphocyte and non-lymphocyte fraction by MACS. Basophils were double stained for CD49b and FcεRIα. Macrophages, dendritic cells, neutrophils, eosinophils, B cells and T cells were stained for F4/80, CD11c, Gr-1, Siglec-F, CD19 and CD3ε, respectively. The respective populations were gated and displayed in histogram plots. The black graph represents Mcpt5-Cre ROSA26-EYFP double transgenic mice, the shaded graph represents the Cre-negative but ROSA26-EYFP-positive control (littermates in most instances)
Absence of significant Cre-mediated genotoxicity in mast cells of Mcpt5-Cre animals
In order to exclude a reduced proliferative potential of mast cells in Mcpt5-Cre transgenic mice due to Cre-mediated genotoxic effects, we compared the percentage of mast cells in peritoneal lavage and the proliferation of peritoneal mast cells from Mcpt5-Cre ROSA26-EYFP double transgenic or wild-type mice in vitro. Cell suspensions obtained by peritoneal lavage contained similar numbers of mast cells in wild-type and double transgenic mice (Fig. 4a). Total peritoneal lavage cells were cultured in the presence of stem cell factor. After 23 days, these cultures contained 98% mast cells as judged by expression of CD117 and FcɛRIα (not shown). By this time, absolute cell numbers had increased more than 200-fold in both cultures (Fig. 4b). Importantly, the mast cells grown from the Mcpt5-Cre ROSA26-EYFP positive mouse uniformly expressed EYFP (not shown) ruling out an overgrowth of the culture by a (hypothetical) minor population of mast cells, which do not express Cre. These results show that Mcpt5-Cre positive mast cells are not growth retarded and therefore do not seem to be significantly affected by Cre-mediated genotoxicity.
Fig. 4Numbers and proliferative potential of peritoneal mast cells in Mcpt5-Cre transgenic mice. (a) Mast cells were quantified in peritoneal lavage fluid from control (n = 9) and Mcpt5-Cre (7 A-Mcpt5-Cre and 3 male X-Mcpt5-Cre) mice. Mast cells were detected as CD117+FcεRIα+ cells by FACS. (b) Total cells from peritoneal lavage fluid from one control and one A-Mcpt5Cre ROSA26-EYFP double transgenic mouse were incubated in complete Opti-MEM supplemented with 4% supernatant from CHO transfectants secreting murine SCF (Malbec et al. 2007). The CHO transfectants were generated by S. Lyman, Immunex, Seattle and were kindly provided by P. Dubreuil. Cell numbers were quantified at each passage every 3–4 days by counting trypan blue-excluding cells
Discussion
Here, we describe a novel Cre transgenic mouse allowing mast cell-specific knock out of genes in vivo. We employed BAC transgene technology to express a Cre cassette under the control of the Mcpt5 promoter. To our knowledge, this is the first report of mast cell-specific Cre-mediated recombination in the mouse.
To date, important insights into functions of mast cells have been gained by reconstitution of mast cell-deficient mice with mast cells differentiated in vitro from gene deficient bone marrow or embryonic and hematopoietic stem cells (Galli et al. 2005b; Grimbaldeston et al. 2005; Kitamura et al. 1978; Tsai et al. 2005). While this model yielded important information, inherent technical problems limit experimentation and interpretation of data in this system (Tsai et al. 2005). We expect that mast cell-specific knock out of genes using our new Cre transgenic line will greatly facilitate investigation of mast cell biology.
The two Cre transgenic lines demonstrate that the Mcpt5-promoter allows efficient and cell type-specific expression of a Cre transgene inserted into the first exon. One animal of the line A-Mcpt5-Cre (characterized by autosomal transgene inheritance) showed non-specific Cre-mediated recombination in non-mast cells while seven animals of this line did not. This finding likely reflects accidental early activity of the transgenic Mcpt5 promoter, e.g. in one stem cell during the embryonic four cell stage. Future analysis will define the frequency of such events more precisely. In females of the X-Mcpt5-Cre line (X-chromosomal inheritance), Cre-mediated recombination occurred in only about half of the mast cells due to inactivation of the transgene-carrying X-chromosome. Incomplete inactivation of particular genes may be advantageous if the attenuation of a severe phenotype is desired.
Cre recombinase has been shown to mediate genotoxicity in some Cre transgenic or Cre knock in mouse strains resulting in a reduced proliferative potential or even death of Cre expressing cells (Schmidt-Supprian and Rajewsky 2007). In Mcpt5-Cre mice, however, we found no difference in mast cell numbers or proliferation of mast cells ex vivo in response to stem cell factor indicating that Cre-mediated genome damage is not a prominent feature of these mice.
We are currently refining our system further to achieve also inducible mast cell-specific gene inactivation in adult mice. In addition, we are breeding the new lines to iDTR mice (Buch et al. 2005) aiming at diphtheria toxin-inducible ablation of the mast cell lineage in adult animals.

mast cells
cre/loxp-mediated recombination
conditional gene targeting
cre transgenic mice


The impact of maternal separation on adult mouse behaviour and on the total neuron number in the mouse hippocampus

The maternal separation paradigm has been applied to C57BL/6J mice as an animal developmental model for understanding structural deficits leading to abnormal behaviour. A maternal separation (MS) model was used on postnatal day (PND) 9, where the pups were removed from their mother for 24 h (MS24). When the pups were 10 weeks old, the level of anxiety and fear was measured with two behavioural tests; an open field test and an elevated plus maze test. The Barnes platform maze was used to test spatial learning, and memory by using acquisition trials followed by reverse trial sessions. The MS24 mice spent more time in the open arms of the elevated plus maze compared to controls, but no other treatment differences were found in the emotional behavioural tests. However, in the reverse trial for the Barnes maze test there was a significant difference in the frequency of visits to the old goal, the number of errors made by the MS24 mice compared to controls and in total distance moved. The mice were subsequently sacrificed and the total number of neurons estimated in the hippocampus using the optical fractionator. We found a significant loss of neurons in the dentate gyrus in MS mice compared to controls. Apparently a single maternal separation can impact the number of neurons in mouse hippocampus either by a decrease of neurogenesis or as an increase in neuron apoptosis. This study is the first to assess the result of maternal separation combining behaviour and stereology.
Introduction
Developing an animal model of mental disorders is controversial due to the human nature of the symptoms such as hallucinations, delusions and poverty of speech. These symptoms can only be adequately assessed by psychological assessments and therefore cannot be modulated in animals. A way to try to circumvent these problems is studying certain psychological or psychophysiological aspects of mental disorders such as latent inhibition, prepulse inhibition or P50 gating (Ellenbroek and Cools 1990, 1995; Geyer and Markou 1995; Ellenbroek et al. 2004). However, it is still unclear how (and if) these abnormalities are linked to the symptoms of mental illness. In rodent models prepulse inhibition (PPI) of the acoustic startle response is a model of sensorimotor gating mechanisms in the brain, while an equivalent reaction in humans is eye blinking (Braff et al. 1978; Ellenbroek et al. 1998). It has been shown that rat pups that underwent 24 h maternal separation on postnatal day (PND) 6 or 9 expressed reduced prepulse inhibition (PPI) on postnatal day 69 and had hyperactivity of the dopamineric system involving the dopamine neurotransmitter system via the hypothalamic–pituitary–adrenal (HPA) axis (Ellenbroek and Cools 1995). Further, the PPI deficits could be reversed with typical antipsychotic drugs like haloperidol and were not detected prior to puberty (Ellenbroek et al. 1998). Due to the changes seen in the HPA axis, the dopamine system, hippocampus and long-term behavioural effects modelling deficits seen in mental patients has lead Ellenbroek and co-workers to hypothesis the 24-h maternal deprivation model to be a “schizophrenia-like” neurodevelopmental animal model (Ellenbroek and Cools 1998, 2002; Ellenbroek et al. 1998, 2004, 2005).
One of the striking characteristics of the developing neuroendocrine stress system in the mouse and rat is a period of reduced stress-responsiveness, the so-called stress hypo-responsive period (SHRP) (Schapiro et al. 1962; Cirulli et al. 1994; Schmidt et al. 2002). From about postnatal day (PND) 4–14 in the rat, and PND 1–12 in the mouse, the animals neuroendocrine system is characterized by a low basal corticosterone level and by the inability of a mild stressor, e.g. exposure to novelty, to induce a corticosterone response (Cirulli et al. 1994; Schmidt et al. 2003). In this study, we used the C57BL/6 inbred mouse. Due to a similar postnatal neurodevelopmental course in the mouse and rat (Clancy et al. 2001), we used PND 9 as the day of separation equivalent to Ellenbroek and co-workers day of choice in the rat. We tested if a 24-h maternal separation on PND 9 can cause an adult phenotype characterized by altered levels of activity and anxiety, learning and memory dysfunction, deficits in behavioural flexibility (reversal deficits) as well as changes in number of neurons in the hippocampus and its subregions in the mouse brain.
Material and methods
Animals
The offspring of 4 male and 8 female C57BL/6J mice (8 weeks old) obtained from Taconic Europe (Taconic Farms Inc., DK) were used in this study. The animals were acclimatized to the animal facility for 1 week. Multiparous females were used, since there is a higher rate of offspring survival. The animals were housed under a 12:12 h light/dark cycle (lights on at 6 a.m.) with constant temperature (21 ± 2°C) and humidity (52 ± 2%) in Macrolon type III cages with environmental enrichment in the form of wood splints bedding (aspen 4HV), wood shavings, 1 piece of Aspen Corner 15, 1 standard mouse house made of recycled cardboard, 1 aspen chewing stick size medium, and pads of nesting material (all obtained from Brogaarden, DK). Food (Altromin pills NR 1324) and tap water were available ad libitum. Two females were placed in a male’s cage for a period of 1 week to ensure conception, followed by separation of the two females to their own cages.
Maternal separation
Pregnant females were checked for litters daily at 09:00 a.m. If litters were found, the day of birth was defined as PND 0 for that litter. On PND 0, litters were randomly assigned to maternal separation (MS) (N = 16, 9 males and 7 females), or to standard facility rearing (SFR). For the behavioural testing, 16 MS animals (4 males and 6 females) and 5 SFR animals (2 males and 3 females) were included, while 12 of the MS animals (2 males and 10 females) and 7 control animals (2 males and 5 females) were used for cell counting. Litters were not culled or sexed at birth to minimize the handling of the pups, but male and female pups were separated at weaning (PND 28) and group housed with their siblings, which resulted in 2–5 mice per cage. The environmental enrichment applied to the mothers was also applied to the MS and control animals.
The 24-h deprivation was carried out on PND 9 starting at 8 a.m. The pups remained in the home cage but were placed in a separate room with the same temperature, humidity and lighting conditions as the home stable. The cage was placed on a heating pad, which had a constant temperature of 31°C. No food or water was available during the separation. The dam was placed in a cage with similar facilities as the home cage in the home stable. The pups were checked every 3 h, using a red light during the night. Body weights were recorded before and after separation. Immediately after 24 h the dams were returned to the home cage and reunited with the pups.
Test for anxiety and fear related behaviour
When the pups reached 10 weeks of age they were subjected once to the open field test (Hall 1934) and the elevated plus maze test, which is based on the procedure used by Montgomery (1955) and later validated by Pellow et al. (1985). Behaviour was analysed using EthoVision (Noldus, Groeningen, The Netherlands). All behavioural testing took place between 10 a.m. and 3 p.m.
Open field test (OFT)
The open field consisted of a circular wooden platform (diameter 90 cm) surrounded by a 43 cm high wall with a camera mounted directly above. A central circle of 31 cm diameter was defined in the behaviour analysis software. Three 60-W light bulbs illuminated the arena. On the day of testing each animal was transported in a cardboard box to the centre of the open field and behaviour was recorded for 10 min. After the trial the maze was cleaned with a solution of acetic acid and soap water and faecal boli were counted.
The following parameters were calculated; total distance moved (cm), time spent in central circle and time spent in peripheral zone (expressed as % of session duration).
Elevated plus maze
The plus maze was elevated 50 cm above the ground and consisted of two opposing open arms (21 cm × 8 cm) connected by a central square (8 cm × 8 cm) to two opposing enclosed arms of the same size with 32 cm high walls. A video camera placed above the maze recorded the animals’ behaviour. On the day of testing, the animal was placed in the centre of the maze and behaviour was recorded for 10 min. Between trials the maze was cleaned as described for the OFT. The following parameters were calculated; total duration (s) in open and closed arms, the number of entries into the open and closed arms and the total distance moved (cm). From these parameters the ratio of entries into the open arms to the total number of arm entries, and the ratio of time spent in the open arms to time spent in both open and closed arms was calculated.
Test for spatial memory; Barnes maze
The Barnes maze (Barnes 1975) consisted of a circular, white-coated platform 90 cm in diameter and elevated 50 cm over the ground. Sixteen 5 cm wide holes were evenly distributed around the perimeter, 2.5 cm from the edge. A pair of rails was placed under two opposing holes to hold the hidden escape box. The escape box was a dark plastic storage box with a 5 cm diameter hole in the lid. A dark cylindrical cardboard tube (7.5 cm × 7 cm high) with a lid was used as the transport and start chamber.
Three 60-W bulbs illuminating the maze and high irregular rock and techno music played from a computer in a random manner provided the aversive stimuli. As with the previous test, a digital video camera mounted above the maze recorded animal’s behaviours.
Shaping
For 2 days before testing commenced, the animals were trained to enter the hidden escape box. Using the transport cylinder, the mouse was placed near the edge of the target hole with the hidden escape box underneath. Two cardboard walls blocked entry to adjacent holes. Only dim lightning and no noise was used during this phase of the experiment. The animal was allowed 5 min to enter the escape box and if this failed, it was placed manually inside. When the animal entered the box it was quickly carried to the home cage.
Acquisition trials
Six consecutive trials were given, one per day. The animal was placed in the transport cylinder, oriented in a random direction, in the centre of the maze. The aversive stimuli were turned on and the cylinder removed. Recording in the behaviour-observation software began immediately after the experimenter had left the room. The trial ended after 5 min or when the animal entered the hidden escape box. If the animal failed to enter the box or re-entered the maze after recording was stopped, the aversive stimuli were turned back on and the animal was allowed 5 min more to enter the escape box. If that also failed, the animal was manually placed in the box. After completion of each trial, the box was placed in the home cage.
Reversal trials
Three days after acquisition trials, the escape box was placed underneath the hole opposite to the hole that had been the target during acquisition training. Reversal training was conducted for seven consecutive days as described above.
Parameters
Each of the 16 escape holes in the Barnes maze was defined as a separate zone-of-interest in the behaviour analysis software. The following parameters were analysed: latency to target [time from start of the trial to first entry into the target hole zone (s)], total distance moved (cm) and error frequency (number of visits to other holes than the target hole). During reversal training two additional parameters were analysed: the mean number of visits to the old target hole over the seven trials (the hole where the escape box was located during acquisition training) and the mean number of visits to the two holes adjacent to the old target hole.
Three different search strategies could be distinguished and were scored manually. The random search strategy is characterized by a non-systematic exploration of the maze with many centre maze crossings and some perseverations. Perseverations were defined as repeatedly searching the same hole or two adjacent holes (Bach et al. 1995). Secondly, there is the serial search strategy, defined as systematic consecutive hole searching in a clockwise or counter clockwise manner and finally a spatial search strategy, defined as searching less than three holes from the location of the goal (Barnes 1979; Bach et al. 1995; Inman-Wood et al. 2000; Zhang et al. 2002; Raber et al. 2004) (Fig. 1).
Fig. 1Three different search strategies could be applied: Random, a pattern that include many hole examinations in a random manner; Serial, a relative systematic search either clockwise or counterclockwise; Spatial, a pattern where the target box is found within relative short time and with high accuracy
Fixation and embedding
Approximately 1 week after all tests were concluded, the animals were scarified by CO2 asphyxiation and within 1 h the brains were dissected out and placed in a 37% formalin solution (Fixatin). The brains were split through corpus callosum, separating the two hemispheres. After systematic random assignment of left or right hemisphere they were coloured on the outer surface to preserve a coded sequence and embedded in paraffin with an automatic vacuum tissue processor (Leica ASP300). The hemispheres were then mounted horizontally balancing on needles and embedded in a paraffin block with up to four hemispheres in one block
Sectioning
The whole hemisphere was cut horizontally with a Leica (model SM 2400) microtome with a microtome setting of 40 μm thickness. All sections were mounted on silicone-coated glass (Frost +). After a minimum of 24 h in a 40°C heating cupboard, the sections were sampled systematic uniformly randomly (SURS). The first section was randomly selected from a random number table, hereafter every 6th section was sampled systematically for staining and counting. This provided a total of 8–10 sections containing the hippocampus per specimen. To be able to account for block advance (BA) the block height was measured for every 100th section. The BA determines the hitting probability of the particles within the block (see later for calculation). Furthermore, the paraffin shrinkage effect was calculated and amounted to about 70%. The sections were stained with a modified Vogt’s Cresyl violet acetate (Armed forces).
Optical design equipment
The optical design equipment consisted of an Olympus BH-2 microscope with an oil immersion 100× objective of high numerical aperture (NA = 1.40), which allows focusing in a thin focal plane inside a thick section. A camera transmits the image to a computer screen where a counting frame is superimposed using the computer-assisted stereological (CAST)-Grid software (Visiopharm, Hørsholm, DK). A motorized automatic stage was used to control movement in the x,y- plane via a connected joystick. Movement in the z-axis was controlled manually with the focus button on the microscope and the distance between the upper and lower surfaces of the sections was measured with a Heidenhein microcator (Heidenhain, Germany) with a precision of 0.5 μm.
Definitions and divisions of the hippocampus
The hippocampus was subdivided into five regions; the dentate gyrus (DG), the hilus of the dentate gyrus (CA4), regio inferior (CA3/2), region superior (CA1) and subiculum.
Neurons were counted manually in the sub-sampled sections containing the hippocampus, using the optical disector design. The layers comprising the hippocampus were defined consistently in all sections using the terminology of Blackstad (1956). It was not considered too difficult to differentiate the neuronal layers in horizontal sections (Fig. 2). The granule cell layer and the pyramidal cell layers also contain the cell bodies of basket cells and glial cells. The glial cells can be easily identified and counted separately, but the basket cells are so similar in appearance to the granule and pyramidal cells that they are included in the estimates. It was previously shown that they comprise less than 1% of the neurons in these layers (review West et al. 1991).
Fig. 2A schematic drawing of the hippocampus with the five subregions identified in this study. Dg, the granule cell layer of the dentate gyrus; h/CA4, hilus of the dentate gyrus; ri, regio inferior; CA3/2, rs, region superior; CA1, s, subiculum
Stereological equations
Estimation of the total neuron number, N
For the estimation of total neuron numbers, neurons were counted in optical disectors and sampled according to the so-called fractionator principles. In a fractionator, cells are counted directly in a known fraction of the different hippocampal subdivisions and the total neuron number, N, is estimated by multiplying the number of particles counted with the reciprocal sampling fractions: where ssf is the section sampling fractions, asf the area sampling fraction, and hsf the height sampling fraction. The bilateral cell number is estimated by multiplying the unilateral number ∑Q− by 2. This is admissible when the right or left hippocampus is sampled systematically randomly.
The asf is known as the area of the counting frame of the disector relative to the area associated with each x,y-step movement of the disector: After having ascertained that the cell density was constant within the disector height, the height sampling fraction was defined as: is the q− weighted mean section thickness (Gundersen et al. 1988; Dorph-Petersen 2001).
Estimation of total volume—the Cavalieri estimator
Besides estimating total neuron numbers, it was also decided to estimate total volume of the different compartments of the hippocampus although the main purpose was estimation of cell numbers. Estimates of volume were obtained according to the principles of Cavalieri’s basic estimator (Gundersen and Jensen 1987) and corrected for shrinkage. Notice that these volumes were not used for the estimation of total neuron number. The estimates of total neuron number obtained by the fractionator design are independent of the containing volume. Further, when total number and volume is estimated, the density, NV (cells/mm3) can be obtained as well without extra work.
Error predictions
The precision of the two different estimates (neuron number and volume) can be expressed by the coefficient of error, CE. The CE for the Cavalieri estimation of volume was first formulated and described by Gundersen and Jensen (1987) revised in Gundersen et al. (1999). Table 1 gives an example of how CE for both volume and neuron estimates are calculated in this study (see also Gundersen et al. 1999).
Table 1Example of how the CE is calculated for neuron number and volume respectivelySectionQj−A:Qi  × QiB:Qi × Qi + 1C:Qi  × Qi + 2SectionPj−A:Pi × PiB:Pi × Pi + 1C:Pi  × Pi + 21111216162311242282563,1361,1761,0642111214433321441399378341612124193613425743993518324541985393963933486113371112117616573996816256240352839615915225330909241081022484132–1052520–11636––11416––Sum1985,5143,4982,583Sum4122313897
When an appropriate number of sections have been chosen (10 or a little less), it is the number of points counted (the noise) which decides the precision of the estimate. To count about 200 points per sample is usually enough to obtain a CE around 5–8%, unless the object is very irregular (Gundersen and Jensen 1987; Gundersen et al. 1988; West and Gundersen 1990). When CE is estimated for the cell counting, Noise is equal to ∑Q−.
Statistical analyses
All data were analysed with the statistical software packages SPSS (Statistical Package for the Social Sciences, version 14.00) or SigmaStat (version 2.0). If Levene’s test for equality of variances did not fail, a t test was applied to test for differences between the two experimental groups. For data where normality tests failed, the non-parametric tests Mann–Whitney U test and Kruskal–Wallis test were applied. The animals performance over the six acquisition trials and the seven reversal trials in the Barnes maze was analysed by mixed-model analysis of variance with trial as within-subject factor and experimental group as between-subjects factor. Homogeneity of the variance-difference scores was determined by Mauchly’s test of sphericity [SPSS Inc. Chicago, IL, USA. SPSS Base version 11.5 User Manual; 2004]. When the assumption of sphericity was violated, degrees of freedom were adjusted with the Huynh–Feldt correction. Group differences in strategy selection in the Barnes maze were analysed with the likelihood-ratio chi-square test for each trial separately. Group differences were considered significant when P < 0.05.
Results
Only significant behavioural results are depicted and statistically elaborated.
Body weights
A significant weight loss was found in the pups after separation (P = 0.019, paired t test). However, 8 weeks after separation the body weights of MS and SFR animals were similar (P = 0.61, unpaired t test).
Open field test
Both experimental groups spent more time in the periphery of the open field than the centre (P < 0.001, t test). There was no significant difference between MS and SFR groups in either distance moved or frequency of visits to and time spent in the centre or periphery of the open field (data not shown).
Elevated plus maze
The MS animals spent more time in the open arms than the SFR animals and allotted a greater percentage of time spent in both open and closed arms to the open arms (P = 0.035 and P = 0.046, t test, respectively; Fig. 3).
Fig. 3The mean (+SEM) time spent in both the open and closed arms. *There was a significant difference between MS 24 (N = 16) and SFR 24 (N = 5) in time spent in open arms (P < 0.05, ANOVA). ** Time spent in closed arms compared with time spent in open arms (P < 0.001, t test)
In trial progress there was a significant difference in both frequency and duration in closed and open arms [Closed arms frequency: (F(9,171) = 3.544, P < 0.0001) and duration: (Wilk’s λ F(9,11) = 6.045, P = 0.004), Open arms frequency: (Wilk’s λ F(9,11) = 8.086, P = 0.001) and duration: (F(9,171) = 6.253, P < 0.0001)].
There was no significant difference in any of the other parameters analysed.
Barnes maze
Acquisition trials
Latency to reach the target hole, total distance moved on the maze, and error frequency all decreased significantly over the course of the six acquisition trials (F(4.3,77.4) = 3.88, P = 0.005; F(5,90) = 2.56, P = 0.033; F(3.9,70.1) = 2.49, P = 0.05; respectively), indicating that the animals indeed learned the task. There was, however, no significant difference between the two experimental groups.
Reversal trials
Latency to reach the new target hole (Fig. 4 left) total distance moved on the maze (Fig. 5), and error frequency (Fig. 6) all decreased significantly over the course of the seven reversal trials (F(3.1;55.95) = 3.08, P < 0.05; F(1.85;33.41) = 16.24, P < 0.001; F(3.43,61.68) = 18.79, P < 0.001, respectively). The MS24 animals made significantly more errors over the course of reversal training (F(1, 18) = 4.79, P < 0.05; Fig. 6) and travelled longer distances (Fig. 5; P = 0.008, Mann–Whitney U-test). This might indicate that the MS animals did not learn the reversal task as fast as the controls.
Fig. 4Left: Mean (±SEM) latency to “Goal”. There was a significant difference in Trial progress and a significant Trial x Group interaction (P < 0.05). Right: Mean (±SEM) frequency of visits to “Old Goal”. There was * a significant difference between the two groups (P < 0.05, Mann–Whitney U test) and a significant decrease over time (P < 0.05, RM ANOVA). MS 24 (N = 16) and SFR (N = 5)Fig. 5Mean (±SEM) distance moved in reverse trials. There was a significant decrease over time (P < 0.05, RM ANOVA) and * a significant difference between the two treatment groups (P < 0.05, Mann–Whitney U test) but no significant Trial x Group interaction. MS 24 (N = 16) and SFR (N = 5)Fig. 6Mean (±SEM) frequency of errors in MS 24 (N = 16) and SFR (N = 5) * there was a significant difference between the two treatment groups and a significant decrease in Trial progress (P < 0.05). MS 24 (N = 16) and SFR (N = 5)
The MS24 animals made significantly more visits to the old target hole (Fig. 4 right) and the two adjacent holes over the course of the seven reversal trials (t(17.5) = 3.72, P < 0.01; t(17.5) = 4.34, P < 0.001).
This tendency to venture to the old goal and the adjacent holes could be an indication of perseveration, a behavioural pattern also found in other animal models and in schizophrenic patients (Bleuler 1950; Bach et al. 1995; review Crider 1997)
Search strategies
The animal’s search pattern evolved over time (Fig. 7). The usual behaviour sequence was a progression from a random to a serial, and finally to a spatial search strategy. This sequence indicates that the animals became more accurate and more efficient in locating the correct hole. Likelihood-ratio chi-square tests showed that the experimental groups differed in their choice of search strategy on days 2 and 3 of acquisition training (χ(2) = 10.5, P < 0.01; χ(2) = 6.6, P < 0.05, respectively). Already at this early time the SFR animals adopted a serial search strategy while the MS animals maintained a random search strategy. On all other acquisition and reversal training days the two experimental groups did not differ significantly with respect to their choice of search strategy.
Fig. 7Overview of mean (%) search strategies applied in the acquisition and reverse trial sessions. The search pattern evolved over time from a random to a serial, and finally to a spatial search strategy. From acquisition days 2 and 3, the SFR animals adopted a serial search strategy, while the MS animals maintained a random search strategy. On all other acquisition and reversal training days the two experimental groups did not differ significantly with respect to their choice of search strategy. SFR (N = 5) animals to the left and MS (N = 16) animals to the right
Neuron number
The stereological sampling is shown in Table 2.
Table 2Overview of the stereological sampling used in this studySubiculumCA4CA1CA3DGMS (maternal separated)Area (frame) (μm2)770–1,160710–1,100259259259Z depth (μm)2020202020X and Y step (μm)17580100100170∑Q−208 ± 36.5140 ± 23.8254 ± 15.7193 ± 10.3205 ± 15.4Mean section thickness, tq− (μm)39.5 ± 0.240.0 ± 0.3039.0 ± 0.338.9 ± 0.3039.3 ± 0.30Height sampling fraction, hsf1.97 ± 0.012.00 ± 0.011.95 ± 0.021.95 ± 0.011.96 ± 0.02Area sampling fraction, asf57.4 ± 11.516.8 ± 4.0638.7 ± 0.0030.7 ± 0.01111 ± 0.00Section sampling fraction (k), ssf66666Guard zone, (μm)5.5–75.5–75.5–75.5–75.5–7SFR (Control)Area (frame) (μm2)1,222 ± 61.61,388 ± 34.7197 ± 0.1240 ± 6.2197 ± 0.1Z depth (μm)2020202020X and Y step (μm)20095100100170∑Q−245 ± 14.7190 ± 14.4215 ± 13.6188 ± 11.8201 ± 7.5Mean section thickness, tq− (μm)39.0 ± 0.6039.1 ± 0.538.5 ± 0.638.2 ± 0.438.6 ± 0.4Height sampling fraction, hsf1.95 ± 0.031.95 ± 0.021.93 ± 0.031.91 ± 0.021.93 ± 0.02Area sampling fraction, asf33.3 ± 1.626.46 ± 0.2950.9 ± 0.0342.0 ± 1.28147 ± 0.07Section sampling fraction (k), ssf66666Guard zone, (μm)5.5–75.5–75.5–75.5–75.5–7
There was no significant difference in the total bilateral neuron number in hippocampus (Table 3). However, in the five subregions of hippocampus, there was a significant difference in the dentate gyrus (P = 0.029, Students t test), but none in the four other regions (see Fig. 8; Table 3). The neuron loss in the dentate gyrus is equivalent to a 20% reduction in the maternal separated animals compared with controls. The results should be interpreted with caution due to the low number of subjects.
Table 3Total estimated neuron number in five subregions of the hippocampusMaternally separated (MS) N = 12SexSubiculumCA4CA1CA3DGTotalSubjectNeurons (103)CENeurons (103)CENeurons (103)CE Neurons (103)CE Neurons (103)CENeurons (103)CEFemale3310.0842.90.132920.061900.076720.071.5290.09Female2640.1034.80.132430.061890.075760.071.3070.09Female3710.0818.60.183030.062260.064740.081.3930.09Female1070.0821.20.081420.081120.093340.107160.09Female1480.0723.10.072130.071440.083540.108820.08Female1320.0720.60.082620.061740.076650.071.2540.07Female1380.0720.20.071550.081630.085520.071.0280.07Female1890.1267.90.132020.071340.084600.081.0530.10Female1440.0523.10.072300.071710.075970.071.1660.07Female1970.0520.90.072290.072030.074840.091.1350.07Male2370.1067.20.122570.061910.074870.091.2390.09Male2420.1037.40.132240.061860.077880.061.4880.09Mean2080.0833.20.112290.071740.075380.081.1820.08CVcontrol (SFR) N = 70.500.360.280.210.260.25Female1860.0726.30.072850.071750.088040.071.4760.07Female1760.0730.20.092400.071910.086580.081.2960.08Female1690.0731.50.072730.072050.076550.081.3330.07Female1940.0525.30.091820.081860.076170.061.2040.08Female1620.0834.10.092780.071990.087250.071.3970.05Male1570.0820.30.092060.081150.096130.091.1120.07Male2210.0628.00.072850.071910.076440.091.3680.07Mean1810.0727.90.082500.071800.086740.081.3120.08CV0.070.120.170.060.110.08Student’s t testP = 0.41P = 0.46P = 0.37P = 0.65P = 0.02*P = 0.20* P < 0.05Fig. 8The total neuron number for the two treatment groups in five sub-regions of the mouse hippocampus for the pooled data in MS (N = 12) and SFR (N = 7). * MS ≠ SFR, P < 0.05, Student´s t test in the dentate gyrus. filled circle MS; open circle SFR; Sub Subiculum, CA 4 Hilus, DG Dentate Gyrus
Volume
After Cavalieri estimation of volume and correction for shrinkage (67.7%) a Student’s t test did not show any significant difference in either the total hippocampal volume or in the five subregions of the hippocampus between groups (Table 4).
Table 4Total estimated volume in five subregions of the hippocampusMaternally separated (MS) N = 12SexSubiculumCA4CA1CA3DGTotalSubjectVolume mm2CEVolume mm2CEVolume mm2CEVolume mm2CEVolume mm2CEVolume mm2CEFemale4.330.050.690.081.460.051.520.051.800.039.790.05Female3.380.050.690.061.140.061.390.051.230.047.840.05Female4.250.050.590.071.540.051.720.041.180.049.290.05Female1.660.120.600.070.520.091.000.061.060.044.840.08Female2.400.090.860.060.790.081.260.051.290.046.590.07Female1.730.120.650.071.070.061.390.051.330.036.170.07Female2.260.100.690.060.680.091.420.051.220.046.260.07Female2.290.071.040.081.000.061.260.051.120.066.710.06Female2.150.070.790.060.910.071.450.051.130.036.430.06Female3.000.060.740.060.980.071.470.051.130.067.320.06Male2.980.061.010.061.070.061.390.051.150.057.000.06Male3.310.050.910.051.220.061.630.042.180.039.250.05Mean2.810.080.770.071.030.071.410.051.320.047.340.06CVContol(SFR) N = 70.320.200.290.130.250.20Female2.970.080.780.071.260.061.690.041.560.038.250.06Female2.290.090.910.071.010.061.220.051.450.036.880.07Female2.510.090.870.061.160.061.880.041.380.047.820.06Female2.550.090.520.090.850.071.300.050.730.046.050.07Female2.100.110.860.070.980.071.480.051.300.046.730.07Male2.320.100.520.101.030.071.190.051.730.056.780.08Male2.830.080.700.071.170.051.460.051.580.047.720.06 Mean2.510.090.750.081.070.051.460.051.390.047.180.07CV0.120.190.130.170.230.11Student’s t testP = 0.40P = 0.79P = 0.76P = 0.62P = 0.66P = 0.79
Discussion
In the present study, 24 h maternal separation on PND 9 in mice pups resulted in a 20% neuron decrease in the dentate gyrus and behavioural perseverations in the Barnes maze. The anxiety and thus stress related behavioural tests conducted did not show any indications of an elevated anxiety level. On the contrary, the MS mice showed a reduced sign of anxiety, since they ventured more often onto the open arms than the equivalent control group. This could indicate that the SHRP was not repressed, in spite of our expectations. The results were thus surprising and contradicted some but not all other findings (Pihoker et al. 1993; Plotsky and Meany 1993; Cirulli et al. 1994; Wigger and Neuman 1999; Boccia and Pedersen 2001; Parfitt et al. 2004). A study by Lehman et al. (1999) found that MS 24 at PND 9 did not result in the anxiety/fear response predicted by the group (Lehmann et al. 1999). Others have also found that effects of a manipulation of the HPA axis cannot always be used to predict effects of the same manipulation of fear/anxiety expression at the behavioural level (see Lehmann et al. 1999; Parfitt et al. 2004). Furthermore, Parfitt et al. (2004) reported that maternally separated C57BL/6 male mice had a prolonged increase in plasma CORT after an acute stressor, but in adulthood showed no increased fear/anxiety behavioural response (Parfitt et al. 2004). The lack of fear response does evidently not necessarily mean that the corticosterone plasma level is not elevated in MS animals but since the corticosterone level was not measured this question remains unanswered. Finally, a study by Francis et al. (2002) concluded that MS rats subjected to an enriched environment could reverse the effect from MS on the HPA function and anxiety behaviour (Francis et al. 2002). In conclusion, we found that the MS mice in our study did not show behavioural indications on a repressed SHRP, which might be explained by the enriched environment the mice were kept in. The corticosterone plasma level was not measured.
In the data presented for the Barnes maze, there were no significant differences between the two treatment groups during the acquisition period. Latency to reach the target hole, total distance moved on the maze, and error frequency all decreased significantly over the course of the six acquisition trials and seven reversal trials, indicating that the animals were able to learn the task with time, which is in agreement with other studies (e.g. Pompl et al. 1999).
One consequence of hippocampal dysfunction is perseveration (Devenport et al. 1988). For the MS animals the group differences in the number of errors made, the goal parameter and the distance moved were therefore of interest. The differences found in the parameter “Close to Old Goal” the significantly higher frequency of “Errors” and visits to “Old Goal” by the MS 24 animals indicate that the MS animals were more perseverant which could point to a hippocampal lesion (Devenport et al. 1988; Bach et al. 1995). However, since perseveration is also a feature of prefrontal cortical dysfunction and there was no hippocampal dysfunction in the acquisition trials, we can only conclude that the behaviour in reversal trial may be related to hippocampus, but we cannot exclude that it is was caused by, e.g. a prefrontal cortical deficit.
One of the advantages of the Barnes maze is its ability to reveal the search strategies applied by the mice. The search strategies can be an indication of how well the cognitive abilities in the mice are due to the use of extra-maze cues (Barnes 1979; Holmes et al. 2002). In this study, an overall search pattern evolved with time. On initial trials the mice tended to use a random pattern and explore many incorrect holes, often returning to the centre after investigating the edge of the platform. With more experience the number of centre crossings decreased and a more systematic search was applied. Thus in later trials many mice went directly to the correct hole or one or two holes away before locating the hidden box. The mice learned the task better after a previous introduction to the platform and used the “Spatial” search strategy earlier. Furthermore, the SFR mice used the search strategy “Serial” significantly faster than the MS mice in the acquisition trials, but this was not true in the reversal trials. The serial search strategy requires the mouse to use the multiple relationships among extra-maze stimuli to find the escape tunnel (Barnes 1979; Bach et al. 1995). So apparently the MS mice needed more time to acquire to the spatial search strategy when learning the task, which could indicate a learning disability. However, since the MS mice did not show difficulties in using the spatial search strategy in the reverse trials, no firm conclusions can be made on this point.
Over 85% of granule cell neurogenesis are previously described to occur postnatally in the rodent with peak neurogenesis between PND 5 and 7 and total cell number increasing throughout the first year and continuing throughout life (Altman and Das 1965; Schlessinger et al. 1975; Bayer et al. 1982; Kuhn et al. 1996; Kempermann et al. 1998). Much research has focused on the neurogenesis in both the adult and newborn hippocampus of mammals (Kempermann et al. 1997; Gould et al. 1997; Eriksson et al. 1998; Tanapat et al. 1998, 2001; Gould et al. 1999; Lemaire et al. 2000; Malberg et al. 2000; Raber et al. 2004; Mirescu et al. 2004; Greisen et al. 2005) but only a few of these studies have used modified stereological methods (Kempermann et al. 1997; Eriksson et al. 1998; Gould et al. 1999; Lemaire et al. 2000; Malberg et al. 2000; Greisen et al. 2005). None of these previous studies have quantified the total cell numbers in the hippocampus using stereology in early trauma animal models or in humans. The model can apparently induce a neuron change in the hippocampus but whether the lower neuron number is a decrease due to a neuron loss or because neurogenesis has been affected in the peak period could not be determined in this study. Secondly, the impact of the neuron loss is not immediately linked to a possible memory deficit. Even though the MS animals had a reduced emotional responds in the elevated plus maze and showed perseverance in the Barnes maze, the cognitive deficits could just as well be due to a prefrontal damage and thus not a result of the decreased neuron numbers in DG.
We tested if a 24-h maternal separation on PND 9 can cause an adult phenotype characterized by altered levels of activity and anxiety, learning and memory dysfunction, deficits in behavioural flexibility (reversal deficits) as well as changes in number of neurons in the hippocampus and its subregions in the mouse brain. We found that a single 24 h maternal separation on PND 9 could elicit a reduced stress response in the elevated plus maze and induce perseveration behaviour in the Barnes maze. Further, a 20% reduction in total neuron numbers was found in the dentate gyrus of the hippocampus.
Ethics
The experiment was carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and the Danish legislation regulating animal experiments (Animal care and housing BEK nr 687 from 25/07/2003). The Danish Animal Experiments Inspectorate approved the protocols (Journal No. 2003/561–781).

maternal separation
behaviour
hippocampus
stereology


Multislice CT angiography in the selection of patients with ruptured intracranial aneurysms suitable for clipping or coiling

Introduction We sought to establish whether CT angiography (CTA) can be applied to the planning and performance of clipping or coiling in ruptured intracranial aneurysms without recourse to intraarterial digital subtraction angiography (IA-DSA).
Introduction
Subarachnoid haemorrhage (SAH) is caused by aneurysmal rupture in 70–85% of patients [1, 2]. In a systematic review, Hop et al. found fatality rates ranging from 32% to 67% [3]. Furthermore, 10–20% of patients remained functionally dependent after SAH. Rapid diagnostic evaluation and treatment are crucial for the patient’s outcome.
Intraarterial digital subtraction angiography (IA-DSA) has been the main technique for detecting and characterizing intracranial aneurysms and remains the gold standard. However, IA-DSA is invasive and time consuming, and carries a risk of neurological complications of 0.5–1.8% with permanent deficit in 0.09–0.5% [4–6]. Serious non-neurological complications, which occur in 0.6% of patients, include groin hematoma, peripheral thromboembolism, transient hypotension and arteriovenous fistulas [4]. Furthermore, IA-DSA may increase the risk of rebleeding [7, 8]. It has been demonstrated that three-dimensional CT angiography (3D-CTA) can reliably detect intracranial aneurysms [9–14]. Only after replacement of IA-DSA by CTA can the advantages of CTA be fully realized in the clinical setting. We report here our clinical experience with both 16- and 64-detector row CTA as the first and intended only diagnostic and treatment decision-making study for intracranial aneurysms in patients with acute SAH.
Materials and methods
Subjects
Between April 2003 and January 2006 all patients presenting with a SAH to the University Medical Centre Groningen consecutively underwent CTA as the first diagnostic study. Based on the CTA findings, patients were selected for surgical clipping or endovascular coiling of a ruptured intracranial aneurysm.
SAH was suspected on clinical grounds and confirmed by unenhanced CT or by blood pigments on lumbar puncture.
Imaging protocols
The CT examinations were performed on a 16- or 64-multidetector row spiral CT machine (Somatom Sensation 16 or 64; Siemens Medical Systems, Erlangen, Germany), based on a standard protocol. The 64-multisclice CT was implemented in the Emergency Department in December 2004.
Parameters for 16-slice CT for diagnosis of aneurysm: via an intravenous cannula in the antecubital fossa, 80 ml of contrast agent (Visipaque 320) was injected with a power injector at a rate of 4 ml/s. Injection of contrast agent was followed by a flush of 50 ml 0.9% saline (Stellant; NaCl Neck Angio) injected at the same rate. A manual fluoroscopic bolus-triggered system, with the internal carotid arteries as reference point and a delay of 4 s, determined the optimal timing. The CTA protocol parameters were as follows: spiral mode, rotation time 0.5 s, reconstruction interval 0.75 mm at Kernel H20, 120 kV/200 mAs, acquisition time 10 s, scan range from the C1 vertebral body to the vertex parallel to the orbitomeatal line.
Parameters for 16-slice CT for diagnosis of SAH: gantry un-angled, spiral mode, rotation time 0.75 s, 16-detector rows at 0.75-mm intervals, table speed 6 mm/rotation, reconstruction interval 3 mm at Kernel H30 and acquisition parameters 120 kV/200 mAs. The actual acquisition time was approximately 15 s.
Parameters for 64-slice CTA for diagnosis of aneurysm: rotation time 1 s, table speed 15.4 mm/rotation, reconstruction interval 0.6 mm at Kernel H20, 120 kV/260 mAs, acquisition time 9 s and scan range extending from the C1 vertebral body to the vertex parallel to the orbitomeatal line. The protocol parameters for contrast agent injection remained unchanged.
Parameters for 64-slice CT for diagnosis of SAH: gantry un-angled, spiral mode, rotation time 1 s, 64 detector rows at 0.6-mm intervals, table speed 9.6 mm/rotation, reconstruction interval 2 mm at Kernel H30, acquisition parameters 120 kV/260 mAs and acquisition time 14 s.
Postprocessing of CTA
Source images were transferred to a remote computer workstation (Odelft Benelux diagnostic imaging) for viewing. Initial careful review of axial images was considered imperative. During this review any areas of concern could be noted. Two-dimensional maximum intensity projection (MIP) views and three-dimensional (3-D) surface-rendered and volume-rendered reconstructions were reformatted from the raw image date on a Vitrea computer workstation by one of the neuroradiologists.
Parameters for IA-DSA and postprocessing
From April 2003 until April 2004 the IA-DSA studies were produced on a digital angiographic unit (Siemens Multiskop with InfiMed image processing) with a 512×512 pixel matrix. From April 2004 onwards the studies were performed on a Siemens Axiom Artis angiographic unit with a 1024×1024 pixel matrix. Selective four- or six-vessel angiography using a standard projection format was performed initially and additional views were obtained if required to identify the parent vessel and aneurysm neck more clearly. The amount of contrast medium (Visipaque 270) used was 8 ml for the internal carotid artery and 6 ml for the external carotid artery, and the injection rate was 6 ml/s when the tip of the catheter was in the internal carotid artery and 3–4 ml/s when the tip of the catheter was in the external carotid artery. The rate of injection into the vertebrobasilar system was 6–8 ml/s to a total amount of 8 ml.
In certain situations, rotational 3-D angiography was performed to better delineate the anatomic details of an aneurysm. Rotational 3-D angiography was performed on a Siemens Axiom Artis angiographic unit. The C-arm rotates in a continuous 200° arc around the patient’s head during a prolonged intraarterial catheter injection of contrast medium (28 ml Visipaque, injection rate 4 ml/s). The raw date images were transferred to a Leonardo workstation (AX Applications) from which 3-D volume-rendered reconstructions were reformatted.
Image review and data analysis
The presence of an aneurysm, its size and morphology, its parent and feeding vessels and the collateral circulation at the circle of Willis were determined by one of the diagnostic or interventional neuroradiologists. If multiple aneurysms were detected, the usual criteria were applied to decide which aneurysm was responsible for the haemorrhage. These criteria included the unenhanced CT findings (distribution of blood) and the size and irregularity of the aneurysm.
All diagnostic findings were discussed with the neurosurgeons. The CTA results were categorized into proven ruptured aneurysm, inconclusive or negative. Patients with a proven ruptured aneurysm were selected subsequently for coiling or clipping. The surgical and endovascular findings were compared to the CTA findings. In general, ruptured aneurysms in the anterior circulation were selected for either coiling or clipping. Ruptured aneurysms located in the posterior circulation were preferably coiled. Giant intracranial aneurysms were preferably treated surgically. A ruptured aneurysm in association with an intraparenchymatous haemorrhage was most often selected for clipping of the aneurysm and surgical evacuation of the haematoma.
Patients categorized as inconclusive or negative underwent IA-DSA. In patients with a perimesencephalic blood distribution, one IA-DSA examination was performed. In patients with a nonperimesencephalic blood distribution a second IA-DSA was performed if the first one was negative. IA-DSA was considered the gold standard. CTA was considered false-negative when IA-DSA revealed a ruptured aneurysm or when rebleeding occurred.
The positive predictive value, negative predictive value, sensitivity, specificity and accuracy of CTA per patient were calculated. The chi-squared test was used to compare the performance of 16-slice CTA and 64-slice CTA for the identification of intracranial aneurysms. Differences with a P value less than 0.05 were considered significant.
The IA-DSA findings in patients in the inconclusive category were compared with the CTA findings to assess whether IA-DSA actually provided any additional information.
Results
Patient population
From April 2003 until January 2006 292 patients with SAH underwent CTA. Excluded from the study were 68 patients, of whom 24 were excluded because of a nonaneurysmal cause of the SAH including trauma (n = 17), arteriovenous malformation (n = 6) and anticoagulant therapy (n = 1), 3 because of hypertension and intracerebral haematoma, 4 because of comorbidity or advanced age, 2 were excluded because of poor clinical condition and 1 because if poor clinical grading and advanced age, and 31 died from the initial effect of SAH, rebleed or vasospasm with ischemia. Two patients refused therapy and one patient was referred to another hospital for treatment.
The study included 224 patients, 89 men and 135 women with a mean (±SD) age of 52.7 ± 10.7 years (range 22–79 years). Their clinical condition just before treatment was classified according to the original Hunt and Hess grading system: 99 patients were classified as grade I, 45 as grade II, 58 as grade III, 20 as grade IV, and 2 as grade V [15].
Detection of intracranial aneurysms
Of the 224 patients, 140 underwent 16-slice CTA and 84 underwent 64-slice CTA. The CTA results were categorized as proven ruptured intracranial aneurysm (133 patients, 59%), inconclusive (31 patients, 14%), or negative for aneurysm (60 patients, 27%).
Positive CTA result
In this category 133 ruptured aneurysms were diagnosed in 133 patients. In 25 patients 32 associated unruptured aneurysms were diagnosed. The distributions of the locations and sizes of the aneurysms are shown in Tables 1, 2, 3 and 4. An overview of the results in this subgroup is presented in Fig. 1.
Table 1Location of symptomatic intracranial aneurysms in 224 patientsAneurysm locationCTA-positive (n = 133)CTA-inconclusive (n = 31)aCTA-negative (n = 60)bCoiling (n = 78)Clipping (n = 55)Coiling (n = 8)Clipping (n = 12)Anterior circulationAnterior communicating artery3425551Pericallosal artery22Middle cerebral artery318161Internal carotid artery3111Posterior communicating artery1781Anterior choroideal artery1Posterior circulationBasilar tip11Vertebral junction1Posterior cerebral artery1Posterior inferior cerebellar artery511Superior cerebellar artery1a11 patients had no proven aneurysm.bFive patients had false-negative CTA, in four of whom a ruptured aneurysm was diagnosed on repeat angiography.Table 2Size distribution of symptomatic intracranial aneurysms in 224 patientsSize (mm)CTA-positive (n = 133)CTA-inconclusive (n = 31)aCTA-negative (n = 60)b < 5471245–970410–1414315–2411≥251a11 patients had no proven aneurysm.bFive patients had false-negative CTA, in four of whom a ruptured aneurysm was diagnosed on repeat angiography.Table 3Location of asymptomatic intracranial aneurysms in 224 patientsAneurysm locationCTA-positiveCTA inconclusiveAnterior circulationAnterior communicating artery6a3Pericallosal artery2Middle cerebral artery11b4Internal carotid artery9aPosterior communicating artery6aPosterior circulationBasilar tip1Junction of vertebral artery1Posterior inferior cerebellar artery1aOne false-negative on CTA.bTwo false-negatives on CTA.Table 4Size distribution of asymptomatic intracranial aneurysms in 224 patients Size (mm)CTA-positiveCTA-inconclusive < 530a45–96310–141aFive false-negative on CTA.Fig. 1Flow chart of CTA results
The majority of patients were treated within 3 days of SAH (n = 99, 75%). Of the 133 CTA-positive patients, 78 (59%) were coiled and 55 (41%) were clipped. Treatment conversion was needed in three patients, in two because of difficult aneurysm morphology and in one because of rebleeding during surgery (dura not yet opened). In two patients treatment conversion was necessary because of incorrect treatment selection based on CTA (Fig. 2).
Fig. 2CTA and IA-DSA results in a 66-year-old woman with SAH Hunt and Hess grade III. CTA showed four aneurysms: an aneurysm of the anterior communicating artery (AComA) and two bilateral aneurysms of the middle cerebral artery and one aneurysm of the pericallosal artery. The aneurysm of the AComA was regarded as symptomatic at the time of initial SAH and its CTA-proven morphology showed both coiling and clipping to be a difficult challenge. Coiling of the AComA aneurysm was tried on the 2nd day. The session was aborted because the neck of the aneurysm was broad and the aneurysm incorporated both A2 segments. Unfortunately, rebleeding occurred after coiling. The morphology of the aneurysm excluded complete occlusion by clipping on the 25th day. In the postoperative course again two rebleedings occurred and the patient died. At autopsy a ruptured pericallosal aneurysm was seen more distal to the clipped aneurysm. a Coronal MIP CTA; b volume-rendered CTA; c AP view DSA, selective catheterization of left internal carotid artery; d volume-rendered IA-DSA; e, f autopsy (red arrow anterior communicating artery aneurysm, yellow arrow middle cerebral aneurysm, black arrow anterior cerebral artery (A2 segment), blue arrow pericallosal artery aneurysm
All ruptured intracranial aneurysms were confirmed by surgery or endovascular treatment. In two patients IA-DSA was performed after surgical treatment for evaluation of coiling of asymptomatic aneurysms.
In four patients (3%) a fatal rebleeding occurred during follow-up, in one patient soon after complete occlusion of the aneurysm with coiling, in one patient on the 5th day after clipping, in one patient 2 weeks after incomplete occlusion of the aneurysm with coiling and in one patient almost 3 years after clipping. In all patients the blood distribution of the rebleeding was the same as that of the primary SAH. Although permission was not granted for an autopsy in two patients, it was considered most probable that the rebleeding was caused by the treated aneurysm. In two patients an autopsy was performed. In one a ruptured pericallosal aneurysm was seen, 1.5 cm more distal from the clipped anterior communicating artery aneurysm which was regarded as symptomatic at the time of initial SAH (Fig. 2), and in the other a haematoma surrounding a prepontine cavernous haemangioma and an endovascular treated dissecting aneurysm of the basilar artery were seen.
The presence of CT-diagnosed additional asymptomatic aneurysms was checked in 22 patients. These patients had 29 aneurysms. Five aneurysms were confirmed at surgery and subsequently clipped, 5 aneurysms were checked with IA-DSA and subsequently coiled and 19 aneurysms were confirmed with IA-DSA. Three aneurysms in three patients were not verified. In four patients five asymptomatic aneurysms were false-negative on CTA. All were smaller than 5 mm. Four aneurysms were diagnosed with IA-DSA during an embolization session, one of them was also embolized. Another aneurysm was considered a vessel loop of the middle cerebral artery on CTA. However, an aneurysm of the middle cerebral artery was seen during surgery of a ruptured aneurysm of the anterior communicating artery. Clipping of the aneurysm of the middle cerebral artery was also performed.
Inconclusive CTA result
In 31 patients IA-DSA was performed because of inconclusive CTA results. The indications for IA-DSA examination are presented in Table 5. An overview of the results in patients in this category is presented in Fig. 1.
Table 5Indications for IA-DSA examination in 31 patientsIndicationNo. of patientsMore information required regarding location and orientationSymptomatic aneurysm10Asymptomatic aneurysm2More information required regarding presence of intraaneurysmal thrombus in symptomatic giant aneurysm2Differentiation between asymptomatic and symptomatic aneurysm1Differentiation between infundibulum, vessel loop and aneurysm5Fisher grade IV SAH1Arterial vasospasm3Discrepancy between diagnosed intracranial aneurysm and distribution of blood1Incomplete angiography of circle of Willis3Overprojection of venous structures1Variance of normal intracranial vessel anatomy1Amalgam artefacts1
In 11 patients (35%) IA-DSA confirmed the results of CTA. In 17 patients (55%) IA-DSA was able to give further diagnostic information required for a correct patient selection for therapy. In two patients (6%) no additional diagnostic information could was obtained from IA-DSA. In both patients vasospasm of a vertebral artery resulted in an inconclusive CTA, but also excluded selective catheterization with IA-DSA. A second CTA was negative in both patients. In one patient (3%) treatment selection was based on a false-positive IA-DSA. CTA was inconclusive because of amalgam artefacts in the region of the right posterior inferior cerebellar artery (PICA). An aneurysm of the right PICA was diagnosed on the first IA-DSA. A second IA-DSA was performed with the intention of coiling. However, with additional views the aneurysm turned out to be a vessel loop.
No ruptured aneurysms were found in ten patients (four nonperimesencephalic SAH, four with perimesencephalic SAH and two negative on unenhanced CT). In 20 patients 20 ruptured intracranial aneurysms and 6 additional unruptured aneurysms were found. In one patient only an asymptomatic aneurysm was diagnosed (Tables 1, 2, 3 and 4). Of the ruptured aneurysms, 12 were clipped and 6 were coiled. Two ruptured aneurysms were clipped after failure of endovascular treatment. Two asymptomatic aneurysms were clipped.
Negative CTA result
In 60 patients CTA was considered negative. An overview of the results in this category of patients is presented in Fig. 1. Of these 60 patients, 13 (22%) had negative findings on unenhanced CT, and 47 (78%) had positive findings on unenhanced CT, and of the latter 30 had a perimesencephalic blood distribution and 17 had a nonperimesencephalic SAH [16]. In 11 (85%) of those with negative findings on unenhanced CT, one IA-DSA was done. In one patient a second IA-DSA was performed and in one patient a second CTA was performed. No rebleedings occurred. CTA was true-negative in all these patients.
In 21 patients (70%) with perimesencephalic SAH, IA-DSA was performed once, and in one of them CTA was repeated once and in one CTA was repeated twice. In eight patients IA-DSA was repeated once and in one patient IA-DSA was repeated twice. In this category CTA was true-negative in all these patients. No rebleedings occurred.
In nine patients (53%) with nonperimesencephalic SAH IA-DSA was performed once, and in two of them a follow-up MRA was done and in one CTA was repeated. In seven patients a second IA-DSA was done. In one patient IA-DSA was repeated twice. In five patients (29%) with nonperimesencephalic SAH, CTA was false-negative (Tables 1 and 2). In one of these patients only IA-DSA was able to detect a 3-mm ruptured aneurysm of the anterior communicating artery, and in the other four a rebleeding occurred despite an initially negative CTA and IA-DSA. Repeat angiography was performed in three patients: a ruptured aneurysm was shown by CTA in two and by IA-DSA in one. One patient died before repeat angiography. Two patients were treated successfully. One patient died before treatment due to the direct effect of the rebleeding. The explanations for false-negative results were interpretation mistakes (two aneurysms recognized retrospectively on CTA and IA-DSA), and haematoma demonstrated on CTA surrounding and compressing the aneurysm leading to interpretation error on both CTA and IA-DSA in one patient and on only CTA in another (IA-DSA showed the aneurysm); the findings were uncertain in one patient.
Statistical analysis
The diagnostic value of both 16- and 64-slice CTA are outlined in Tables 6 and 7. A comparison of the results of 16-and 64-slice CTA is presented in Table 8. No statistically significant differences were found.
Table 6Diagnostic value of CTA in ruptured aneurysmsDiagnostic valueTrue positive132 patientsFalse positive1 patientTrue negative55 patientsFalse negative6 patientsPositive predictive value99%Negative predictive value90%Sensitivity96%Specificity98%Accuracy96%Table 7Diagnostic value of CTA in additional aneurysmsDiagnostic valueTotal number detected with CTA25 patients (32 aneurysms)Presence checked22 patients (29 aneurysms)True positive22 patients (29 aneurysms)False positive0True negative120 patientsaFalse negative4 patients (5 aneurysms)Positive predictive value100%Negative predictive value97%Sensitivity85%Specificity100%Accuracy97%aIncluding 60 CTA-negative patients and 60 CTA-positive patients. Of the CTA-positive patients, 44 were not examined with IA-DSA as standard control.Table 8Comparison of results of 16- and 64-slice CTA for detection of intracranial aneurysms 16-slice (n = 140 patients)64-slice (n = 84 patients)CTA resultPositive7459Negative4515Inconclusivea2110Ruptured aneurysmsTrue positive7359False positive10True negative4213False negative4b2Positive predictive value (%)99100Negative predictive value (%)9187Sensitivity (%)9597Specificity (%)98100Accuracy (%)9697Unruptured aneurysmsTotal number on CTA12 (15 aneurysms)13 (17 aneurysms)Presence checked11 (14 aneurysms)11 (15 aneurysms)True positive11 (14 aneurysms)11 (15 aneurysms)False positive00True negative75c45dFalse negative1 (1 aneurysm)3 (4 aneurysms)Positive predictive value (%)100100Negative predictive value (%)9994Sensitivity (%)9279Specificity (%)100100Accuracy (%)9995aPatients with an inconclusive result were not included in the statistical analysis.bIncluding one CTA-positive patient.cIncluding 45 CTA-negative patients and 30 CTA-positive patients. Of the CTA-positive patients, 31 were not examined with IA-DSA as standard control.dIncluding 15 CTA-negative patients and 30 CTA-positive patients. Of the CTA-positive patients, 13 were not examined with IA–DSA.
Discussion
Our primary aim was to assess whether CTA is useful clinically in planning and performing clipping or coiling, especially in the acute phase in ruptured intracranial aneurysms, without recourse to IA-DSA. We demonstrated that it was possible to treat more than half of all patients with a ruptured intracranial aneurysm using only CTA. By avoiding conventional angiography, it was possible to streamline the management of ruptured aneurysm during the acute phase. Further, 3D-CTA was able to help in deciding whether to clip or to coil; in only two patients was treatment conversion needed due to incorrect treatment selection based on CTA.
We found 3D-CTA to be a simple, reliable, quick and minimally invasive imaging modality that reduces the risk of complications caused by conventional angiography and reduces the delay between the patient’s arrival at the hospital and treatment, leading to diminished rebleeding. Matsumoto et al. analyzed the rate of rebleeding of ruptured aneurysms during CTA and conventional angiography, and found 0% (none of 160 patients) for CTA and 1.5% (5 of 317 patients) for conventional angiography [17]. In patients with a ruptured aneurysm and intracerebral haemorrhage CTA saves time when aiming for a fast clot removal. Another advantage is that the radiation dosage is low compared to IA-DSA (1.0 mSv at 200 mAs with the CTA Siemens Sensation 16 and 1.8 mSv at 380 mAs with the CTA Siemens Sensation 64 compared with 3.5–6.5 mSv with conventional angiography). Furthermore, the cost of CTA is one-fourth that of conventional angiography.
Several other studies assessing whether CTA may serve as the sole imaging method for the preoperative work-up of patients with ruptured intracranial aneurysms have been published [17–26]. An overview of these previous studies is presented in Table 9. There is a wide variation in the percentage of patients who have had their symptomatic aneurysms treated based on CTA. This may be influenced positively by the very high aneurysm prevalence and the subsequent very low negative rates of CTA in some studies [22, 23, 25]. In other studies patients with a negative CTA were not enrolled at all [17, 20]. In general, a mean of 15–20% negative angiographies after SAH is accepted [27]. The present study showed a high negative rate for CTA. This may reflect the good awareness of the diagnosis SAH in first-line and second-line health-care and the good access to CTA when the diagnosis SAH is considered. Furthermore, the wide variation in CTA-based treatment may be partially explained by differences in hardware and software used by each group, the rate of technical failures in performing CTA, scanning parameters set for screening the circle of Willis and more peripheral vessels, the experience and scrutiny of the neuroradiologist evaluating each CTA and the willingness of the neurosurgeon and neurointerventional radiologist to rely on CTA alone in each individual case.
Table 9Presentation of previous studies and present studyStudyNo. of patientsCTA-positiveCTA-negativeCTA inconclusive or no CTA-based treatmentTotal patientsCTA-based treatmentTrue-positive CTATotal patientsTrue-negative CTATotal patients218746 (55%)44 (96%)44 (100%)15 (17%)6 (60%)26 (30%)2210988 (81%)87 (99%)87 (100%)5 (5%)5 (100%)16 (15%)238462 (74%)62 (100%)62 (100%)7 (8%)0 (0%)15 (18%)199045 (100%)45 (100%)45 (100%)––45 (50%)1815061 (41%)61 (100%)60 (98%)a24 (16%)24 (100%)65 (43%)2512040 (27%)40 (100%)40 (100%)13 (11%)13 (100%)67 (56%)247827 (35%)27 (100%)27 (100%)20 (26%)20 (100%)b31 (40%)1710093 (93%)93 (100%)93 (100%)––7 (7%)209687 (91%)87 (100%)86 (99%)2––9 (9%)266144 (72%)44 (100%)44 (100%)15 (25%)14 (93%)2 (3%)Present study224133 (59%)133 (100%)132 (99%)260 (27%)55 (92%)31 (14%)aOne false-negative and one false-positive ruptured aneurysm in one patient.bIn five patients with perimesencephalic SAH, IA-DSA as the gold standard control was not performed.
In the present study CTA was false-negative in 8% of patients. The risk of rebleeding after a negative initial CTA was 7%. All false-negatives were in patients with a nonperimesencephalic blood distribution, giving a false-negative rate of 29% and a risk of rebleeding of 24%. It seems unlikely that the false-negative rate of initial CTA and the risk of rebleeding despite a negative initial CTA in patients with a nonperimesencephalic SAH might be influenced negatively by the use of CTA as the first diagnostic tool. Firstly, in all patients with a rebleeding, repeat IA-DSA was also false-negative. Secondly, repeat angiography with CTA performed after a rebleeding still demonstrated an aneurysm.
Furthermore, the findings of other studies using IA-DSA as the first diagnostic tool were similar. In the study by Urbach et al. in 67 patients with a negative initial angiogram after SAH, four ruptured aneurysms were revealed by repeat angiography [28]. Three patients presented with a nonperimesencephalic SAH and one presented with a perimesencephalic SAH. In the study by Bradac et al., 60 of the 440 patients presenting with spontaneous SAH had a negative angiogram [29]. A second angiogram performed 1–4 weeks later revealed a ruptured aneurysm in 5 of the 40 patients. Of these patients, 3 had a second SAH. In all patients a nonperimesencephalic blood distribution was seen on CT.
Because in the present study some aneurysms could be correctly identified retrospectively, we suggest that if, under strong clinical suspicion of a ruptured aneurysm, the CTA is reported as normal, the study should be reviewed by a second neuroradiologist before proceeding to repeat angiography. It is essential to perform a review of axial raw source images. Next, we recommend repeat CTA or IA-DSA when the initial CTA is negative in patients with a nonperimesencephalic SAH. There is no consensus about the time interval for repeat angiography. In practice, the guideline is to repeat angiography after several days to months. The substantial risk of rebleeding in patients with an aneurysmal pattern of haemorrhage in the present study indicates that some cerebral aneurysms are occult on initial CTA. Several factors may explain this finding. Most importantly, there is a learning curve in assessing aneurysms on CTA. Pedersen et al. reported an increase in sensitivity from 88% to 94% after 1 year’s experience [30]. Small aneurysms can be missed when using CTA. CTA had a sensitivity of 50% for aneurysms <2 mm in the study of Wintermark et al. [13]. Distal pericallosal and PICA aneurysms can be missed when restricting the area of coverage to the proximal circle of Willis [31–34]. Thrombosis of the neck of the aneurysm or of the entire sac is another possible reason [23]. Perianeurysmal blood or haematoma may reduce lesion conspicuity [34]. Aneurysms may be mistaken for vascular infundibula (persistent fetal nonaneurysmal dilatation of the proximal vessel) of the posterior communicating or anterior choroidal artery origins if a vessel cannot be identified arising from them [35]. Aneurysms may masquerade as tight vascular loops if the MIP thickness is wide (>3 mm) [34]. In patients with multiple intracranial aneurysms large aneurysms may obscure smaller ones on the CT reconstruction [33]. Aneurysms close to bone (e.g. carotid siphon, ophthalmic and posterior communicating artery) may be overlooked when relying on surface-rendering and volume-rendering techniques or using MIP with bone editing [32, 34, 36–38]. Aneurysms located within or close to the cavernous sinuses are easy to overlook unless thin-section axial and coronal MIP images are reviewed on a slightly wider window width [9].
In patients with a perimesencephalic SAH the chance of finding a posterior fossa aneurysm is low: 2.5–5% [39, 40]. Nonaneurysmal perimesencephalic haemorrhage carries no risk of vasospasm and rebleeding and has been shown to follow a benign course with an excellent prognosis [41]. The chance of finding an aneurysm in 5% of patients has to be weighed against the risk of complications from angiography imposed upon the remaining 95% of patients. CTA has a high accuracy for diagnosis of vertebrobasilar aneurysms and of intracranial aneurysms in general [9–13, 42]. In the present study, in patients with a perimesencephalic SAH and a negative initial CTA, no rebleedings occurred and CTA was true-negative in all. Similarly, in the prospective study of Huttner et al., 69 patients with a perimesencephalic SAH had a negative initial CTA and IA-DSA [43]. A repeat IA-DSA was performed in 38 patients (55%). None of the repeat IA-DSAs showed any additional distinctive features with respect to the first IA-DSA. It therefore seems practical and safe to perform CTA as the first diagnostic tool and to omit repeat angiography if CTA is negative. A formal decision analysis based on these observations confirmed that a strategy where CTA is performed and not followed by conventional angiography, if negative, results in a better utility than a strategy of CTA followed by conventional angiography or of conventional angiography as primary investigation [44].
According to the results of the present study, it seems important to distinguish the two patterns of SAH on CT. The CT criteria of perimesencephalic bleeding have been defined [40]. Different data show that experienced radiologists can accurately discriminate between a perimesencephalic and nonperimesencephalic SAH [12, 40, 45]. Early CT within 3 days is necessary for reliable assessment of the pattern of haemorrhage [12, 40, 46].
A criticism of this study might be that patients treated with endovascular coiling underwent IA-DSA as part of the endovascular procedure and thus should not be counted in the analysis of efficacy of the prospective protocol. However, a shift in management of ruptured intracranial aneurysm from surgery to endovascular treatment has appeared [47]. Endovascular treatment is replacing clipping. The use of CTA as the initial investigation for cerebral aneurysms may offset some of this increased workload whilst also improving workflow.
In conclusion, in this evaluation of the use of 16-row and 64-row multislice CTA in the management of ruptured intracranial aneurysms, we demonstrated that CTA can be used as the first-line diagnostic modality for the management of SAH patients. In CTA-negative patients IA-DSA provided no or marginal added value. IA-DSA is not needed in patients with negative CTA and classic perimesencephalic SAH. Repeat IA-DSA or CTA should still be performed in patients with a nonperimesencephalic SAH, due to false-negative CTAs and IA-DSAs in this patient group. The remaining true indication for IA-DSA was in patients with an inconclusive CTA result. In more than half of those IA-DSA provided relevant new diagnostic information.

intracranial
aneurysm
cta
subarachnoid haemorrhage