Intrathecal implantation surgical considerations in rodents; a review

Highlights

• Intrathecal implantation in rodent is a challenging preclinical surgical practice.

• Sustainable preoperative and intraoperative procedures are defined.

• Major intrathecal implantation procedures in rodents are presented.

• Common side effects of the implantation are presented.

Abstract

Intrathecal access in humans is a routine clinical intervention. However, intrathecal access is limited to drug delivery purposes in rodents, and intrathecal implantation is not a common surgical practice. Preclinically, we have successfully adopted different intrathecal implantation surgical methods for different implant materials in rodents. However, employing the appropriate intrathecal implantation method is a challenging process for surgeons, which includes several steps such as preoperative evaluations and postoperative care. The aim of this review is to define and compare the major documented surgical approaches applicable for intrathecal implantation in rodents along with the associated side effects, as well as highlighting the critical preoperative and postoperative considerations. Overall, this review will provide surgeons with the principles of intrathecal implantation approaches applicable for different implant materials.

Introduction

Intrathecal drug delivery is clinically advantageous over oral or intravenous administration for reasons, such as maximized local drug dose (Ineichen et al., 2017). Anatomically, the intrathecal space in the rat is located between the arachnoid and the pia mater of the spinal cord. It contains cerebrospinal fluid, spinal nerves, and blood vessels (Offermanns and Rosenthal, 2008). In humans, intrathecal access is a well-defined and routine clinical exercise (Deer and Jason, 2011) performed for different indications. These range from purely mechanical purposes, as in lumboperitoneal shunts (Wang et al., 2007), to spinal anaesthesia (Hocking and Wildsmith, 2004), to versatile therapeutic indications such as intrathecal pumps for pain management (Knight et al., 2007) and intrathecal chemotherapy (Olmos-Jimenez et al., 2017). In rodents, while intrathecal access can be performed via different surgical approaches, the principal indication is for drug delivery, as in rats (Yaksh and Rudy, 1976, Hinton et al., 1995) or mice (Hylden and Wilcox, 1980).

From the preclinical experimental point of view, the intrathecal space in rodents is of anatomical importance due to its proximity to key spinal cellular signalling sites, for example, the dorsal horn of the spinal cord in neuropathic pain pathogenesis (Johnston et al., 2004, Wang et al., 1997). As such, critical molecular mediators are elevated in the intrathecal space associated with maintaining health and presentation of disease (Arman et al., 2020). We have previously utilized this ideal anatomical proximity for optimum measurement of key mediating molecules using different materials functionalized as biosensing devices with different intrathecal surgical approaches (Zhang et al., 2019, Arman et al., 2020). However, there are also other experimental potentials, such as the application of neuroelectronic interfaces in the spinal cord (Jackson and Zimmermann 2012). For these indications, a tailored and robust intrathecal implant approach in rodents is a significant experimental need that this review will cover.

Thus, to establish a sustainable intrathecal approach in rodents, we have identified three critical steps of preoperative, intraoperative, and postoperative cares and optimal procedure. Therefore, this review aims to highlight the critical presurgical factors that surgeons should consider before intrathecal implantation in rodents. This will be followed by a review of the established surgical intrathecal access methods in rodents for drug delivery applicable for intrathecal implantation. Finally, we will also highlight the common side effects of intrathecal access in rodents that could be observed in intrathecal implantation procedures.

Anatomically, the meninges in three layers of the pia (innermost), the arachnoid (middle), and the dura (outermost) envelop the spinal cord. The pia in different layers is directly in contact with the spinal cord and separated from the arachnoid layer with cerebrospinal fluid (CSF)-containing subarachnoid space and intrathecal space. The space also contains numerous arachnoid trabeculae, basically the extensions between the pia and arachnoid. The dura includes irregularly arranged collagen fibres containing blood vessels and nerve fibres (Chrubasik et al., 1993).

To access the intrathecal space, given the surgical limitations and specifically the lack of intra-operative guidance we have for rodents, anatomical considerations are probably the most important factor that must be considered before designing an intrathecal implantation experiment. This factor is especially critical if the intervention is a recovery and not terminal and normal behaviour of animals is expected. Obviously, care should be taken to select rats of the same age and weight, if a uniform intrathecal anatomical target is desired.

To have a standardized intrathecal sampling site, the animal anatomical specification needs to be considered. Of importance is that the length of the spinal cord depends on factors such as body weight, sex and strain of animal. As an index, to access the cranial border of lumbar enlargement from the atlanto-occipital joint in male Holtzman rats weighing 350–400 g, 8.5 cm length of polyethylene-10 tubing catheter (PE10) was used. This was confirmed by post-mortem dissections (Yaksh and Rudy, 1976).

In rodents, preoperative care is not applied in different known intrathecal drug delivery methods that will be discussed in this review. However, in more invasive spinal access such as disc puncture models, preoperative care could be applied and is limited to intramuscular buprenorphine for analgesic effects (Fujioka et al., 2016). In another study that employed laminectomized catheterization in Sprague Dawley (SD) rats along with parallel experimental abdomen implantations, postoperative tetracycline (500 mg/l in water) was prescribed to minimize the infection risk (Hinton et al., 1995). Prophylactic oral antibiotic,sulfamethoxazone-trimethoprim (200 mg/5 ml), was also administered in a non-lamictomized catheterization method (Jasmin and Peter, 2001).

Despite the numerous reports of infection following intrathecal implantation procedures in humans (Staats, 2008; Follett et al., 2004), infection complication following intrathecal access is not commonly reported in rodents. Nevertheless, aseptic surgical methods are employed in studies utilizing intrathecal access in rodents (Hinton et al., 1995, Jones and Tuszynski, 2001, Ray et al., 2011). On the other hand, the exclusion of animals with infection symptoms from experiments in intrathecal access models is also reported in studies (Kaya et al., 2019).

Overall, in order to conduct a successful and ethical preclinical intrathecal experiment, it is essential to predict and design appropriate postoperative veterinary care according to the complications expected from the studies. For example, if the intrathecal implantation method involves significant surgical procedures as in Yaksh and Rudy (1976) or Hinton et al. (1995) methods which will be discussed later in this review, clinical wound management (Langlois, 2004) and prophylactic antibiotic therapies, as discussed before, are recommended. For less invasive methods like acute needle puncture, monitoring animals for pain signs and administration of analgesics in rodents such as buprenorphine (02–0.05 mg/kg, Subcutaneous (SC)) is recommended (Curtin et al., 2009).

Regardless of the method of intrathecal access, which will be discussed later, the confirmation of the presence of the drug in the target anatomical location could be performed by different methods. Here, other than the classic tail-flick (Mestre et al., 1994) or the CSF aspiration (Vassal et al., 2015), the most applicable and routine methods include the preliminary studies with the injection of visible dyes such as methylene blue (Papir-Kricheli et al., 1987, Hylden and Wilcox, 1980), Evans blue (Asato et al., 2001) or radiolabel diffusion (Yaksh and Rudy, 1976). These methods could be employed routinely to locate the catheter tip along with post-mortem gross neuroanatomical evaluation in a terminal fashion. The lidocaine paralysis test (10 μl of 2% lidocaine) can also be applied to verify the catheter placement by monitoring hindlimb paralysis (Chen et al., 2012, Hara et al., 2020) in a non-terminal fashion.

In addition to the mentioned method, application of the trail radiographs (dorsal and sagittal) to identify the implanted material (Long et al., 1988a, Long et al., 1988b, Yaksh and Rudy, 1976) is of a superior advantage, especially if the implant is radiopaque.

Chronic caudal introduction of an indwelling polyethylene cannula (PE10) via the atlanto-occipital membrane was first described by Yaksh and Rudy (1976) with the main aim of having a permanent pathway to introduce pharmacologically active solutions in the spinal subarachnoid fluids repeatedly. This procedure starts under anaesthesia with male Holtzman rat (350–400 g) mounted on the stereotaxic instrument followed by dissecting and freeing superficial and then neck muscles from the occipital crest. The authors report using a 20-gauge needle hub that is fixed to the skull with four screws (at Bregma, Lambda and occipital bones) and epoxy cement with the exteriorized end of the catheter fixed in the needle. The PE10 catheter then smoothly inserts into the cranial margin of lumbar enlargement at the thoracic 12 (T12) level. This method is one of the principal methods developed in rodents for intrathecal drug delivery and has been widely used for monitoring behavioural outcomes of drug effects as well as in the evaluation of spinal mediators involved (Malmberg and Yaksh, 1993, Yamamoto and Yaksh, 1993, Akerman et al., 1982).

There are some important considerations with regards to this method. First, the described method could be altered by using different needle gauges fitting into different polyethylene tubes (e.g., 30 gauge needle and PE10 (Belur et al., 2021)). Also, given the mutagenicity of resin cements, relevant recommendations of handing resin mixtures as well as a replacement possibility, including using natural rubber (Yu et al., 2018), may be considered. Furthermore, this method permits repeated access at any timepoint into any selected level of the spinal subarachnoid space in the rat, as in studies in which the lumbar area is accessed (Bogacka et al., 2020). This potential to target a specific anatomical location precisely is of importance. For example, in monitoring dynamic molecular changes in the CSF, such as in studies to target spinal cord lumbar enlargement (Zhang et al., 2019).

Several studies have used the atlanto-occipital approach to deliver drugs intrathecally in rats (Wang et al., 2021, Wang et al., 2021, Ryu et al., 2021, Piotrowska et al., 2021). However, to address the complications, including tissue damage, discussed later in this review, several modifications to this method have been discussed. For example (Buerkle and Tony, 1996) injected pharmacological agents intrathecally with a modified method. Excluding the primary dissection steps mentioned in the original work by Yaksh and Rudy (1976), this method limits the surgical procedures to an incision to the atlanto-occipital membrane followed by insertion of PE10 tube to the intrathecal space. Furthermore, the modified method of Yaksh and Rudy (1976) was introduced as Chronic Dural Port (CDP) for CSF collection in the mouse. Authors recommend this method to apply chemical biosensors intrathecally in free moving and unanesthetized mice to monitor the CSF neurochemicals dynamic changes (Tsuneo et al., 2021).

Overall, despite being a standard and established method for drug delivery, this method is only recommended to meet unique experimental needs, including placement of implant rostral to caudal or implantation in the thoracic area.