Cases
Altogether, 54 pigs were studied; of these, 32 contributed ventricular volume and survival data to this report (Table 1), as follows: (A) Untreated, kaolin-induced hydrocephalic pigs (n=8) survived 19-56 (median 35) days post-kaolin-induction to an age of 51-91 (median 69) days. (B) Shunt-treated hydrocephalic pigs (n= 9) received VP shunts 3-44 (median 22) days after kaolin induction and survived 5-31 (median 28) days post-shunt. Six (6) of these shunted animals contributed neuroimaging data to the untreated hydrocephalic group from MRI scans performed a few days prior to shunting. Endoscopic treatments were performed on 5 hydrocephalic pigs, one with ETV alone, 3 with ETV+CPC, and 1 with CPC alone.; 3 of the ETV+CPC cases were terminal procedures for the development of the technique. Controls included 6 sham/saline-injected pigs that survived 83-90 (median 84) days and 4 intact/non-injected animals that underwent neuroimaging at the approximate pre-kaolin age, 28 or 35 days of age.
Post-induction results – kaolin obstruction and ventriculomegaly
Kaolin deposits consistently formed a solid cast within the basal cisterns (pontine, cerebellopontine angle, interpeduncular, and prepontine) and occasionally within the cistern of the lamina terminalis (Figures 1-3). Kaolin particles were rarely found within the cistern magna and never within the 4th- or 3rd-ventricles, the cerebral aqueduct, or the lateral ventricles. The basal cistern deposits could be identified on MRI throughout the post-induction and post-treatment survival periods (i.e. up to 69-days post-kaolin) on T2-weighted MRI, and were accompanied by arachnoiditis and adherence to the dura mater or membrane of Liliquist. In about ½ of cases, thin, patchy kaolin deposits were found in the subarachnoid space surrounding the base of the hypothalamus and the pituitary gland. No kaolin deposits entered the parenchyma or any of the cerebral ventricles. The basilar artery and its branches were completely embedded in the kaolin cast.
Bilateral ventriculomegaly occurred in all cases used in this report. All portions of the cerebral ventricles expanded, especially the lateral, 3rd and 4th ventricles (Figures 1-3). It is noteworthy that olfactory ventricles were present normally in the domestic pig brain, each with a narrow channel connected to the frontal horn of each lateral ventricle; both the olfactory ventricle and the connecting channel expanded post-kaolin. The cisterna magna always remained open and enlarged, with a thin membranous anterior wall that appeared to separate it from the 4th ventricle. Prominent CSF flow voids, indicative of high pulsatility, were conspicuous in the foramina of Monro, 3rd ventricle, and anterior cerebral aqueduct. The cerebral hemispheres remained gyrencephalic with relatively little distortion of the sulci and gyri. In contrast, the inferior wall of the temporal lobe became extremely thin in response to the considerable expansion of the temporal horn of the lateral ventricle.
Although 85.4% (41/48 cases) of kaolin injections produced ventriculomegaly, the extent of enlargement was highly variable. Less extensive kaolin deposits were present in the basal cisterns in the 3 cases that did not develop ventriculomegaly. One of these pigs received 3 kaolin injections over a 3-week period and never exhibited ventriculomegaly.
Volumetric assessments revealed the ventriculomegaly that occurred post-induction in all cerebral ventricles (i.e. olfactory, lateral, 3rd, and 4th ventricles; Figures 4-6). Quantification of ventricular volume
confirmed the extent and variability of ventriculomegaly (Figure 5). Ventricular volume increased significantly (p<0.001) in all ventricles compared to both intact and sham controls. Regional variations existed within the cerebral ventricles; proportionally, the lateral ventricles expanded the most, followed by the 4th ventricle. All 14 untreated cases exhibited lateral and total ventricular volumes that were above the 2nd standard deviation. This threshold was used to determine which cases were “hydrocephalic”. Linear regression revealed highly significant correlations between lateral ventricular volume and olfactory, third, and fourth ventricle volumes (Figure 6).
Post-induction neurological and cognitive outcomes
For about 24-hours following kaolin injections and recovery, pigs were mildly lethargic with most of the cases exhibiting increases in body temperature (103.5-108.0 C0; normal = 100.5-103.5 C0) and some showing mild ataxia (imbalance, wider stance, stretching of hindlimbs). When this acute response occurred, it usually was resolved within 2-3 days with appropriate medical management (Buprenorphine/Buprenex, Tylenol suppository, Carprofen/Rimadyl, Baytril, and Dexamethasone) and occasional alcohol baths for hyperthermia. Past 2-3 days post-kaolin, these animals did not exhibit any signs of pain or discomfort, and were alert with normal reflexes, consumed food and water, and gained weight throughout the post-kaolin period, which usually continued for about 10-50 days. Nevertheless, neuroimaging revealed ventriculomegaly in most of these animals.
Preliminary results from cognitive testing (Figure 7 and Supplemental Figure 1) showed a non-significant trend toward a higher Recognition Index and significantly more exploratory time (p=0.008) in the post-induction animals compared to normal (pre-induction) pigs, suggesting learning impairments; i.e. more time was needed to become familiar with the novel object (Table 2).
Post-induction success and complications
A total of 48 intracisternal kaolin injections provided data on achieving any amount of ventriculomegaly, as evaluated by post-induction MRI. Overall, the success rate was 75.0% after one injection attempt (36/48; 2 survivors were unsuccessful and 3 died within 24 hours of injection) and 85.4% after one or two injections; the second injection was usually given 1-2 weeks after the first. On the second attempt, 5 cases were successful and 2 were not. In one case, a third injection was unsuccessful. When ventriculomegaly failed, follow-up MRI usually revealed a total or partial lack of kaolin visualized in the basal cisterns.
In addition to the few neurological deficits that occurred acutely after kaolin-induction, several surgical complications arose. Occasionally several penetrations were needed before accessing the cistern magna; at times the occipital bone was contacted first and/or blood was aspirated into the spinal needle. When this occurred, the angle of the needle was either changed slightly but not withdrawn completely and “walked” down the occipital bone, or the needle was removed and a new percutaneous trajectory attempted. These complications occurred in our first few cases, but we quickly learned that optimal injections could be performed if the skin was penetrated in the midline 2-3 cm posterior to the nuchal crest and oriented perpendicular to the neck. It was very important to have an assistant flex the head with the animal in the lateral recumbent position in an effort to open the gap between the occipital bone and the first cervical vertebra. During this maneuver, care must be taken to maintain a patent airway with the endotracheal tube. It also helped to have an assistant observe the needle trajectory from the head of the animal and guide the surgeon performing the injection. Finally, once the kaolin injection has been performed and the equilibration period completed, detachment of the tube extender from the spinal needle should reveal pulsatile extrusion of clear CSF mixed with kaolin from the needle hub; this confirms that the needle tip remained in the cisterna magna during the injection.
Post-shunt results – catheter placement and ventriculomegaly
Our experience with ventriculoperitoneal shunt procedures includes a total of 21 cases performed on pigs 2-44 days after kaolin-induction. This large range was caused either by delays in achieving ventriculomegaly or by the availability of operating rooms. In most cases (n=9, Table 1), shunting was performed 15-25 days post-kaolin. In all cases, CSF was observed within the ventricular catheter as it exited the skull, confirming that the lateral ventricle had been accessed. Post-shunt neuroimaging demonstrated that the catheter was located within the lateral ventricle, and ventriculomegaly was reduced (Figures 1 and 2). In earlier cases, while attempting different trajectories (including coronal) based on pre-shunt neuroimaging, the tip of the catheter occasionally penetrated the head of the caudate nucleus, the thalamus, the hippocampus, or the periventricular white matter. Subsequently, when the insertion of the catheter was limited to 3.5 mm from the external surface of the skull to the approximate location of the foramen of Monro, more consistent placement within the lateral ventricle was achieved (Figures 1D”, 2A”, and 2B”), with the drainage holes open to CSF. In many cases, at least a portion of the catheter contacted the ventricular wall or the choroid plexus (Figures 1D”, 2B”, and 3B”). Contact with the choroid plexus was occasionally associated with conspicuous hemorrhage throughout this structure, and in these cases blood was present in the distal valve and catheter. On two occasions, the plastic anchor, which had been sutured to the occipital bone and the catheter as it exited the skull, had become detached, presumably by traction from the distal catheter as the pig grew. This detachment allowed the ventricular catheter to migrate, in one case dorsally into the parietal and occipital cerebral cortex; in the other case, the catheter had exited the cranial cavity and the proximal tip was located within the cervical musculature. The time course of these catheter migrations was not known. Importantly, both of these animals were asymptomatic for the entire 30-day post-shunt survival period and exhibited reduced ventriculomegaly, suggesting that CSF drainage had been effective for at least a portion of the treatment period.
Post-shunt results – complications
The most prevalent complication was suboptimal placement of the ventricular catheter, especially in the initial cases. Unfortunately, it was difficult to sense tissue penetration as the catheter was advanced; therefore, it was necessary to rely on the pre-shunt MRI for trajectory planning. As we gained experience, several parameters became very important for optimal placement of the ventricular catheter: (1) The dorsoventral ‘height’ of the lateral ventricle should be at least 6.0 mm for a standard catheter with an outside diameter of 2.3 mm; we were most successful when this dimension was 10-13 mm. (2) Insertion should be no more than 3.5 mm from the surface of the occipital bone; this placed the proximal tip of the catheter near the foramen of Monro and prevented penetration of the caudate nucleus. (3) Insertion should be parallel to the dorsal surface of the skull; the tendency to advance perpendicular to the face of the occipital bone caused the catheter to be angled inferiorly with penetration of the thalamus, caudate nucleus, and/or hippocampus. (4) A plastic anchor, screwed into the occipital bone, should be used to secure the ventricular catheter in place; the Medtronic anchor supplied with standard catheters was ideal for this purpose.
All shunted animals recovered well and were asymptomatic for at least 5- and at most 31-days post-shunt (median 25.5). All cases that were terminated electively at 28-31-days post-shunt had functional drainage systems. Shorter post-shunt periods were characterized by acute onset of neurological symptoms; often demise began 6-24 hours before euthanasia was required, and in two cases animals that appeared normal developed profound seizures just 8-10 hours later. No animals with standard VP shunts became febrile or developed infections.
Post-endoscopy results (ETV+CPC, ETV, and CPC)
To date, 5 pigs with hydrocephalus have been treated with neuroendoscopic procedures. Three of these were terminal pilot studies; one pig underwent a combined ETV+CPC and survived for 24 days, while another underwent CPC alone and survived for 10 days. In all cases, the lateral ventricle was accessed unilaterally and the foramen of Monro and choroid plexus visualized readily (Figure 8 and the Supplemental Figure 2 video). In the ETV+CPC case, the ETV was performed first followed by cauterization of as much choroid plexus as possible (Figure 8B -D), and neuroimaging at 3- and 23-days post-treatment revealed further enlarged lateral ventricles, partially cauterized choroid plexus on the side of the endoscopy, and residual blood along the lateral walls of the lateral and third ventricles (Figure 9).
Post-endoscopy results – complications
While the number of cases is limited, one complication may be the accumulation of intraventricular blood. In the CPC case, persistent bleeding from the initial choroid plexus cauterization obscured the anatomy of the third ventricle such that the planned ETV was aborted. In the ETV+CPC case, coagulated blood accumulated along the lateral walls of the lateral and third ventricles; it had diminished by 23-days post-treatment but was still present (Figure 9 B & D). Ventriculomegaly was also persistent; the lateral ventricles remained the same size in the CPC case, and were enlarged further in the ETV+CPC case. The neuroendoscope track had widened considerably and may have even communicated with the cortical subarachnoid space.