Background

Historical Background of the Model

Aneurysmal subarachnoid hemorrhage (SAH) is a severe disease. Concerning their chronological order, the consequences of pathophysiological changes following SAH are often divided into early and delayed effects of the bleeding. Several experimental designs in different animal models exist to investigate these effects of early brain injury and delayed effects of cerebral vasospasm (CVS) due to SAH on neurological deterioration [1–3].

In 1979, the induction of SAH has been firstly described in a rat model [4]. It has been further refined into vessel disruption and blood injection models [5–7]. Concerning blood injection models, methods that include single- or double-injection strategies have been reported [5, 7]. However, the capability of rodents to rapidly clear blood in the subarachnoid space presents a difficulty in single-injection models [8]. Concerning single-injection models, the development of CVS has been described with a peak less than an hour after injection and with a second peak after day one or two [5, 9, 10].

Therefore, a double hemorrhage technique was introduced to better mimic delayed effects of SAH.

Materials and Methods

After its introduction, several technical modifications have been reported concerning the rat double hemorrhage model. To give a valuable overview, we performed a systematic review of the literature.

MEDLINE was searched for published studies based on the rat double hemorrhage model. The keywords “experimental subarachnoid hemorrhage,” “double injection model,” and “rats” were used. Identified studies from the MEDLINE search were further evaluated for inclusion in the systematic review. Two reviewers independently extracted data (V.B. and P.S.). Full-text versions were obtained from all studies that were considered to be potentially relevant by both reviewers. The methods of the used models had to be described in detail. Any disagreement between the reviewers concerning article inclusion or exclusion was resolved by consensus of a third author (E.G.).

Results

We included 20 studies based on the rat double hemorrhage model [9, 11–29]. Details on included studies and technical modifications are given in detail in Table 1. In the following description of the double hemorrhage rat model, several technical considerations from included studies of the review of the literature are described complementary to our own experiences.

Table 1 Results from the systematic review concerning the rat double hemorrhage model
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Animals

Adult male Sprague–Dawley or Wistar rats, usually weighing 225 to 450 g, are commonly used for the double hemorrhage model [7, 17, 22, 24].

Anesthesia, Analgesia, Monitoring, and Perioperative Care

Intraperitoneal application of midazolam (1 mg/kg body weight) and ketamine (100 mg/kg body weight) is used for anesthesia because it appears to be safe and practicable [17, 22, 25, 30–32]. Sedated with this medication, animals are able to breathe spontaneously and sufficient. Furthermore, circulation parameters are stable, and a derangement of cerebral blood flow (CBF) is not observed using this medication [33]. Nevertheless, intubation and mechanical ventilation have been suggested in order to ensure stable physiological conditions [34]. On the other hand, optimal physiological conditions in intubated and mechanical ventilated rodents might retain several acute effects of SAH induction in these animals. However, there are several modifications reported concerning anesthesia: intraperitoneal application of ketamine and xylazine [11, 12, 14–16, 18, 23, 26], chloral hydrate [9, 13], pentobarbital [20, 29], or isoflurane via an endotracheal tube [19, 21, 28] (Table 1).

During the procedure, the body temperature of the animals should be maintained at approximately 37 °C, most commonly by a heading pad. Several other parameters are used to monitor animals during the procedure: middle arterial blood pressure [9, 11, 13, 19–21, 28], systolic blood pressure [24, 25], heart rate [13], paO2 and paCO2 [13, 19–21, 24, 25, 28], hematocrit [21, 25], or blood glucose [21]. An overview of physiological parameters is provided in Table 2.

Table 2 Physiological parameters
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After induction of SAH, application of 5 ml crystalloid solution and 0.0125 mg Fentanyl subcutaneously is recommended. In order to provide sufficient analgesia, animals should receive 5 ml crystalloid solution and 0.0125 mg Fentanyl subcutaneously twice a day during the following observation or treatment course.

SAH Induction

After onset of anesthesia, the left femoral artery is punctuated and a tube (Portex® polythene tube, luminal diameter, 0.96 mm) is inserted for measurement of blood gas values, circulation parameters, and as source of the later injected autologous blood. For additional medication or infusions, another tube is inserted in the left femoral vein. Both tubes are plugged after irrigation with saline infusion, stored under the skin, and left inside the vessels until the animals are sacrificed (Fig. 1).

Fig. 1
figure 1

a Preparation of the femoral vein (arrow) and femoral artery (arrow head). A portex tube is inserted into the femoral artery (hash mark) for measurement of blood gas values, for control of blood pressure, and for blood sample withdrawal. b The femoral vein is cannulated with another tube (asterisk) for administration of additional medication or infusions

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After insertion of both tubes into the vessels and stable conditions, animals are positioned in a stereotactic frame [9, 11–13, 21, 22, 24, 25].

A medial incision is performed after infiltration of the skin and the muscles with local anesthesia (e.g., mepivacaine 1 %). Skin incision should cover the suboccipital region and the arch of C1 (Fig. 2a). Acromiotrapezius muscle is cut strictly along the midline to avoid bleeding. Afterwards, the suboccipital region, the atlanto-occipital membrane and the C1 arch are exposed in a stepwise fashion. After careful surgically disclosure of the atlanto-occipitale membrane, a tube (Portex® polythene tube; luminal diameter, 0.28 mm) is inserted in the cisterna magna (Fig. 2b/c) [22, 24, 25]. Using this tube, 0.1 ml of cerebrospinal fluid is withdrawn. Thereafter, 0.2 ml autologous arterial blood from the femoral artery approach is injected into the cisterna magna through the suboccipital tube to induce the SAH. Takata et al. reported the use of 0.5 ml autologous arterial blood during the initial SAH induction, and 0.3 ml for the second SAH induction [21]. Furthermore, the injection of heparinized venous blood or of a CSF-/blood-mixture has been reported [11, 13, 18]. Additionally, various time ranges for the blood injection have been described: 1 [12], 2 [20, 29], 3 [15, 16, 28], and 10 min [9, 18, 21]. To ensure an optimal subarachnoid distribution of the administered autologous blood, animals are then brought in a head down position and kept in this position for approximately 15 min. Nevertheless, other time periods for optimal subarachnoid distribution of the administered blood have been reported: 5 [13] and 30 min [14–16].

Fig. 2
figure 2

Preparation of cisterna magna: suboccipital skin incision (a), the nuchal muscle layers were divided in the midline and the atlanto-occipital membrane (asterisk) was exposed (b). After insertion of a catheter into the cisterna magna (arrow), 0.1 ml CSF is withdrawn followed by injection of 0.2 ml autologous arterial blood to induce SAH (c)

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The identical surgical procedure was then repeated 24 h after initial SAH for induction of the second SAH [11, 13, 19, 22–26, 28]. Several other reports suggested a second SAH 48 h after initial SAH [9, 12, 14–16, 18, 20, 21, 29]. For the second SAH induction, several other groups reported the use of a reduced amount of blood compared to the initial SAH induction [19, 21, 28].

Treatment and/or Observation Course After SAH Induction

During the following treatment or observation course, several changes in physiological parameters are expected and have been investigated. Table 3 provides an overview concerning the expected pathophysiological parameters in the rat double hemorrhage model.

Table 3 Expected pathophysiological parameters
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Possible outcome measures include neurological assessment with investigation of motor deficits [12, 22, 36]. Furthermore, several radiological investigations were established using digital subtraction angiography (DSA) or magnetic resonance imaging in order to detect and/or monitor CVS and consecutive impairment of cerebral perfusion [5, 17, 22, 37]. In order to verify morphological damage after SAH, histological analyses were performed for the rat double hemorrhage model [17, 24]. Considering the more distinctive sensitivity of the hippocampus and the adjoining cortex areas, vital neurons were counted to depict CVS-related cerebral ischemia.

Furthermore, functional investigations of cerebral arteries were performed to analyze possible drug effects on relaxation or contractibility of cerebrovascular structures [25, 27, 38, 39].

Technical Considerations

An initial small skin incision assures a decrease of surgical trauma and therefore reduction of stress to the animals. The use of a flexible polythene tube for injection of autologous blood into the cisterna magna might minimize the risk of brainstem injuries. The leakage of injected arterial blood can be avoided by adding fibrillar haemostypticum patches (Tabotamp®) and cotton. To avoid collateral damage, injection of autologous blood itself should be carried out slowly with injection volumes no greater than 0.25 ml.

Advantages and Limitations of the Model

Investigation of pathological consequences of aneurysmal subarachnoid hemorrhage is a multifactorial challenge involving consideration of early and delayed effects on neurological deterioration.

When dividing pathological changes after SAH into early and delayed effects, several authors stated that the rat double hemorrhage model seems more eligible to imitate delayed effects of SAH [6, 31, 35, 40] (Table 4). Regarding the emulation of early effects of SAH, perforation models seem to be more suitable [31]. However, concerning the subarachnoid blood distribution, a great variation in amount of subarachnoid blood in injection and perforating models has been reported [35, 40]. While reflecting the clinical setting, great variation of subarachnoid blood volume might lead to the need of larger experimental groups to retain the standard protocol. Furthermore, it has been reported that only a fraction of the previously injected autologous blood was found intracranially when compared to other models [35, 40]. As mentioned above, this might be controlled by adding fibrillar haemostypticum patches to avoid leakage and therefore to better steer the amount of blood injected into the cisterna magna (Fig. 3). The reported high mortality rate in the rat double hemorrhage model has been criticized, but also indicates the induction of severe experimental SAH and is comparable to the clinical situation in humans.

Table 4 Advantages and limitations
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Fig. 3
figure 3

Basal view of the brain in a sham-operated animal (a). Distribution of the blood in the subarachnoid space demontrated by basal view of the brain after induction of SAH (b)

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Nevertheless, besides some limitations the rat double hemorrhage model is a feasible, effective, and customizable rodent model for experimental SAH (Table 4).

First, the experimental setting is cost-effective [41], manageable, and can be established in most centers. As mentioned above, this model provides a good imitation of the clinical setting and time course of delayed effects of CVS in humans suffering from SAH. Eventually, the degree of severity and characteristics of CVS as well as the reduction of CBF are more pronounced in the rat double hemorrhage model when compared to single injection models [6]. As mentioned above, development of CVS was reported shortly after injection and after day one or two in single injection models [5, 9, 10]. In contrast, maximal CVS onset has been reported on day 5 or day 7 after initial SAH in the double hemorrhage model [6, 22]. Despite the rats capability for rapid clearance of blood after subarachnoid hemorrhage, the second SAH induction during the conduction of the double hemorrhage models seem to secure an adequate amount of periarterial blood for longer time period compared to single injection models [6].

Furthermore, the overall mortality rate in double hemorrhage models has been described as high as 50 % [17, 22, 24, 25]. This might even be increased by further invasive diagnostic procedures, e.g., DSA. As described before, death mostly occurred immediately after SAH induction or within 6 h [31]. Nevertheless, the high mortality rate in the double hemorrhage model seems to indicate the successful induction of a severe experimental SAH.

In conclusion, the double hemorrhage model is a well established rodent model to simulate the delayed effects of SAH, and to investigate the use of drugs on morphological ischemic, functional, and delayed consequences of SAH.