Fetal Endoscopic Third Ventriculostomy Is Technically Feasible in Prenatally Induced Hydrocephalus Ovine Model

Abstract

BACKGROUND: 

Congenital obstructive hydrocephalus generates progressive irreversible fetal brain damage by ventricular enlargement and incremental brain tissue compression that leads to maldevelopment and poor clinical outcomes. Intrauterine treatments such as ventriculo-amniotic shunting have been unsuccessfully tried in the eighties.

OBJECTIVE: 

To assess if prenatal endoscopic third ventriculostomy (ETV) is feasible in a large animal model and optimize this technique for ventricular decompression and potential arrest of fetal brain damage in fetal lambs.

METHODS: 

We generated hydrocephalus in 50 fetal lambs by injecting a polymeric agent into the cisterna magna at midgestation (E85). Subsequently, 3 weeks later (E105), fetal ETV was performed using a small rigid fetoscope. The endoscopy entry point was located anterior to the coronal suture, 7 mm from the midline.

RESULTS: 

We obtained clear visualization of the enlarged lateral ventricles by endoscopy in the hydrocephalic fetal lambs. The floor of the third ventricle was bluntly perforated and passed with the scope for a successful ETV. Total success was achieved in 32/50 cases (64%). Causes of failure were blurred vision or third ventricle obliteration by BioGlue in 10/50 (20%) cases, anatomic misdirection of the endoscope in 5 (10%) cases, 2 cases of very narrow foramen of Monro, and 1 case of choroid plexus bleeding. If we exclude the cases artificially blocked by the polymer, we had a successful performance of prenatal-ETV in 80% (32/40) of hydrocephalic fetuses.

CONCLUSION: 

Despite the inherent difficulties arising from ovine brain anatomy, this study shows that innovative fetal ETV is technically feasible in hydrocephalic fetal lambs.

ABBREVIATIONS:

BA

basilar artery

BPD

biparietal diameter

CP

choroid plexus

CPC

choroid plexus cauterization

EP

entry point

ETV

endoscopic third ventriculostomy

FETV

fetal endoscopic third ventriculostomy

FM

foramen of Monro

HCP

hydrocephalus

INJ

injection

IR

infundibular recess

ITA

interthalamic adhesion

LLV

left lateral ventricle

LVD

lateral ventricular diameter

MB

mammillary body

MM

mammillary

RLV

right lateral ventricle

TC

tuber cinereum

TSV

thalamostriate veins.

Congenital obstructive hydrocephalus (HCP) entails a progressive distention of the cerebral ventricular system, caused by a build-up of cerebrospinal fluid (CSF) generated by a blockage fruit of a complex interaction of genetic and environmental factors during fetal development.¹ Without treatment, this can lead to irreversible brain alterations and death in severe cases.² Nowadays, HCP is often diagnosed before birth through routine prenatal ultrasound and fetal magnetic resonance imaging. It is currently treated postnatally with a ventriculo-peritoneal shunt or endoscopic third ventriculostomy (ETV), which reroutes the blocked fluid.^3,4

Increased intracranial pressure within the ventricular system damages the developing brain progressively.^5,6 In theory, fetuses with isolated HCP might benefit from ventricular CSF drainage and intrauterine decompression of the brain mantle around their enlarged ventricular system.

The first attempts to prenatally treat HCP in humans started almost 40 years ago,^7-13 but the results were not completely successful and typically failed to improve outcomes, largely because subjects with associated malformations were not excluded from those trials.^7-13

Currently, ETV is the chosen postnatal treatment in many hydrocephalic neonates and infants.¹⁴ Given this premise, prenatal intrauterine neuroendoscopic intervention to decompress the fetal ventricles, without implanting any additional device, could potentially become the primary treatment for some congenital isolated HCP cases.¹³ Isolated HCP without associated genetic anomalies includes congenital aqueduct stenosis, Chiari II malformation with myelomeningocele, and Dandy-Walker syndrome.¹⁵

The aim of this study, using this HCP-induced fetal lamb model that we recently described and validated,¹⁶ was to test the feasibility of fetal ETV and optimize this technique as an initial way for future translation to human clinic.

METHODS

Animal Husbandry

This study was performed between the period of 2014 to 2019 in 58 fetal lambs from a total of 45 young pregnant ewes (younger than 2 years) with single and twin pregnancies, obtained from the farm at Jesus Uson Minimally Invasive Surgery Centre, Caceres, Spain.

Animals were housed at 22°C and a standard light/dark cycle of 12/12 hours with access to standard sheep chow and drinking water ad libitum.

The experiments followed the guidelines for animal research and were approved by the Institutional Animal Care and Use of Laboratory Animals Committee (IACUC: ES100370001499) at the Jesus Uson Minimally Invasive Surgery Centre, Caceres, Spain.

First Pilot Study in Non-HCP Fetal Lambs

We used 8 normal fetuses from pregnant ewes at 100 to 110 days' gestation with normal not enlarged lateral ventricles, measuring an overall of 3.76 ± 0.05 mm of diameter on the prenatal ultrasounds. We used these animals to try visualization in the worst case scenario and to test different endoscopic instruments.

The first was a sialoscope, and the instrument we finally selected was a 7.3 to 8 Fr ureteroscope (Karl Storz. Ref. 27002 KPK ureteroscope autoclavable, length 25 cm) with 3.6 Fr working channel.

HCP Model Development

We generated HCP in 50 fetal lambs by injecting a polymeric agent, BioGlue, into the cisterna magna at midgestation at E85, as previously described by Oria et al.¹⁶ In brief, maternal laparotomy assisted transuterine or open injection of 1.2 and 2 mL of the polymer into the fetal cisterna magna after obtaining at least 1 to 2 mL of CSF in the aspiration with a 22-gauge needle. We obtained more severe HCP affected fetuses using 2 mL of Bioglue than with the smaller volume.

Ultrasound Monitoring

Using ultrasonography with a transabdominal convex transducer (C5-2 at 2-5 MHz, Philips ATL HDI 5000), all fetuses were monitored before and after injections into the cisterna magna to verify the enlargement of the lateral ventricles. Measurements were performed in the true axial plane in the atria of the lateral ventricle and the glomus of the choroid plexus (CP), as previously described.¹⁶ The lateral ventricular diameter was measured from the inner margin of the medial ventricular wall to the inner margin of the lateral wall, the biparietal diameter was measured from the more external parietal bone point in one side to the contralateral one, and finally, the ratio between the lateral ventricular diameter and the biparietal diameter was calculated based on observed measurements.

HCP severity was calculated using the Cincinnati Hydrocephalus Severity Scale, recently described by our group¹⁶ (Supplementary-data 1, https://links.lww.com/NEU/D580).

Fetal ETV

Twenty days after the BioGlue injection into the cisterna magna in the HCP group, including moderate and severe cases, fetal ETV was performed (E105-110) under maternal general anesthesia and in sterile conditions using 1 of the 2 small instruments and telescopes (Figure 1A). According to the intraoperative observations, we decided transuterine (without open uterine wall) or open-uterus method for neuroendoscopic intervention. The endoscopy entry point was located 5 mm in front of the coronal suture at the midpoint level of the eyelid, about 7 mm away from the midline on the right side of the fetal cranium (Figure 1B). The endoscope was inserted into the frontal horn of the right lateral ventricle (RLV) after a sharp trocar penetrated the skull (Figure 1C and 1D).

FIGURE 1.: Fetal ETV in hydrocephalus animal model. A, Sequence of images showing small surgical instruments for fetal ETV; B, location of the endoscopy entry point (ET) in an E80 fetal lamb; C and D, trocar insertion with exteriorized fetal head. ETV, endoscopic third ventriculostomy.

The different steps of the procedure were visualized on the monitor once the neuroendoscope was introduced into the ventricles (Video). After the ventriculostomy was completed, the endoscope was removed, and the uterus was introduced back into the abdominal cavity.

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Statistical Analysis

Data are expressed as mean and standard deviation, and P-values of <.05 were considered statistically significant (Supplementary-data 2, https://links.lww.com/NEU/D580). We used the GraphPad Prism 9 package for graph and statistical calculations.

RESULTS

Ventricular Dilatation After Cisterna Magna Polymer Injection in Fetuses

We observed fetal brain lateral ventricular dilatation by ultrasonograms soon in the first week after the agent was injected into the cisterna magna at E85 (Table 1) (Supplementary-data 3, https://links.lww.com/NEU/D580). According to the Cincinnati Hydrocephalus Severity Scale, the fetuses injected with BioGlue acquired moderate and severe HCP at 20 ± 5 days after injection (E105) (Supplementary-data 3, https://links.lww.com/NEU/D580).

TABLE 1. - Fetal ETV Successful Rates in HCP Sheep Model

Groups	No. of fetus	Rate (%)	LVD		BPD		LVD/BPD
			E85 INJ	E105 ETV	E85 INJ	E105 ETV	E85 INJ	E105 ETV
Control	8		3.06 ± 0.19	3.56 ± 0.26	30.92 ± 1.48	45.56 ± 2.13	0.01 ± 0.004	0.07 ± 0.004
HCP + Unsuccessful ETV	18	36.00	3.07 ± 0.36	11.13 ± 3.76	33.87 ± 1.23	45.72 ± 6.43	0.09 ± 0.01	0.23 ± 0.05
HCP + Successful ETV	32	64.00	2.95 ± 0.44	10.16 ± 3.34	34 ± 19 3.38	46.48 ± 5.36	0.087 ± 0.01	0.21 ± 0.06

BPD, biparietal diameter; ETV, endoscopic third ventriculostomy; HCP, hydrocephalus; INJ, injection; LVD, lateral ventricular diameter.

Fetal ETV for Moderate and Severe HCP

We attempted fetal ETV in 2 different hydrocephalic subgroups in fetal lambs at E105. One subgroup developed severe HCP (n = 28) and another subgroup developed moderate HCP (n = 22) (Table 2) (Supplementary-data 4, https://links.lww.com/NEU/D580).

TABLE 2. - Fetal ETV Successful Rates in HCP Sheep Model With All the Animals

Groups	No. of fetus	Rate (%)	LVD		BPD		LVD/BPD
			E85 INJ	E105 ETV	E85 INJ	E105 ETV	E85 INJ	E105 ETV
Control	8		3.06 ± 0.19	3.56 ± 0.26	30.92 ± 1.48	45.56 ± 2.13	0.01 ± 0.004	0.07 ± 0.004
HCP + Unsuccessful ETV	18	36.00	3.07 ± 0.36	11.13 ± 3.76	33.87 ± 1.23	45.72 ± 6.43	0.09 ± 0.01	0.23 ± 0.05
Moderate HCP	6	27.27	3.10 ± 0.12	6.40 ± 0.45	33.82 ± 1.82	37.08 ± 0.22	0.09 ± 0.01	0.17 ± 0.01
Severe HCP	12	42.86	3.10 ± 0.14	12.30 ± 0.99	34.25 ± 0.35	49.35 ± 2.62	0.09 ± 0.01	0.25 ± 0.01
HCP + Successful FETV	32	82.05	2.95 ± 0.44	10.16 ± 3.34	34 ± 19 3.38	46.48 ± 5.36	0.087 ± 0.01	0.21 ± 0.06
Moderate HCP + FETV	16	76.19	2.93 ± 0.49	7.27 ± 1.07	33.66 ± 3.73	44.14 ± 6.63	0.09 ± 0.09	0.17 ± 0.03
Severe HCP + FETV	16	88.89	2.96 ± 0.41	13.10 ± 1.90	34.73 ± 3.01	48.50 ± 5.39	0.09 ± 0.01	0.27 ± 0.04

BPD, biparietal diameter; ETV, endoscopic third ventriculostomy; FETV, fetal endoscopic third ventriculostomy; HCP, hydrocephalus; LVD, lateral ventricular diameter.

Operatory Findings and Technical Details

Intraventricular Anatomy in Nonhydrocephalic Fetal Lambs

The entry point for the endoscopy, as we mentioned in the Methods, was located right from the midline of the fetal cranium. The endoscope was inserted into a very narrow normal RLV after the skull was punctured with a sharp trocar. Access into the lateral ventricle by roof was challenging because of minimal normal lateral ventricle diameter as humans. Access was achieved into the intraventricular system and the anatomic landmarks identified although it was laborious. However, visualization was limited because of narrow intraventricular anatomy including lateral ventricles, foramen of Monro (FM), and third ventricle (Figure 2A-2F).

FIGURE 2.: Endoscopic fetal ETV in nonhydrocephalic animal model. A-F, Endoscopic video images showing the fetal ETV in nonhydrocephalic fetal sheep. In this endoscopic intervention, visualization was limited because of narrow intraventricular anatomy including lateral ventricles, foramen of Monro, and third ventricle. CP, choroid plexus; ETV, endoscopic third ventriculostomy; FM, foramen of Monro; MB, mammillary body.

Intraventricular Anatomy and Fetal ETV in Hydrocephalic Fetal Lambs

To visualize the anatomy of the fetal lamb's brain, we performed an intraventricular neuroendoscopy using a small zero-degree rigid 7.3 Fr tip, 25-cm length, ureteroscope (27002 KPK, Karl Storz) at 105 days of gestation, 20 days after the fetal lambs have been injected with an agent to induce fourth ventricular blockage and obtain enlarged lateral cerebral ventricles. The fetal lamb cranium and brain have longer front-to-rear lengths compared with those of humans, but the supratentorial and infratentorial structures, including the brainstem and cerebellum, are on the same horizontal axis.

The entry point for the endoscopy was located on the right side of the fetal cranium with the mentioned references of 5 and 7 mm (Figure 1B). The endoscope was inserted into the frontal horn of the RLV after the skull was punctured with a sharp trocar (Figure 1C and 1D) penetrating the frontal lateral ventricular roof.

The lateral ventricles of the fetal lamb were enlarged and often we observed septum pellucidum disruption as the result of induced severe HCP (Figure 3A and 3B). CP, thalamostriate, and septal veins were identified as landmarks in the ventricles (Figure 3C and 3D). In case of blur fluid or minimal bleeding from the insertion site or trauma in the CP, flushing small volume of Ringer-lactate through the irrigation channel with a 5-mL syringe could improve visualization. This was not completely efficient when we found polymerized BioGlue invading the lateral or third ventricle. When the endoscope was directed into the third ventricle through a very narrow foramen Monro (while protecting the fornix) (Figure 3E), we found this ventricle consistently not dilatated compared with the lateral ventricles because of a compact and large interthalamic adhesion (Figure 3F) that is characteristic of lamb anatomy.

FIGURE 3.: Endoscopic fetal ETV in HCP animal model. Sequence of images showing the fetal ETV in hydrocephalic fetal sheep. The endoscope was inserted into the frontal horn of the RLV. A and B, Lateral ventricles were very enlarged. Septal defect as the result of induced severe HCP were observed. The LLV was also visible because of septal defect. ETV, endoscopic third ventriculostomy; HCP, hydrocephalus; RLV, right lateral ventricle. C, CP in the right ventricle, D, CP and thalamostriate veins (TSV) in the LLV. Anatomic landmarks were identified once into the ventricles. CP, choroid plexus; LLV, left lateral ventricle. E, Direction through a very narrow foramen of Monro (FM). F, Once the third ventricle was entered, the very large Interthalamic adhesion (ITA), G, the left and right mammillary bodies (MM), the IR at 12 o'clock, and the bluish color tuber cinereum (TC) at the floor of the third ventricle were identified. H-K, The floor of the third ventricle at the tuber cinereum (TC) region between the mammillary bodies (MB) and IR was bluntly punctured and perforated. IR, infundibular recess. L, Through the opening in this membrane, we accessed the prepontine cistern, the interpeduncular cistern, and the interpeduncular Liliequist membranes. BA, basilar artery.

The floor of the third ventricle was carefully inspected for the adequate point for the fetal ETV in the tuber cinereum region between the mammillary bodies and the infundibular recess (Figure 3G). This location was bluntly perforated with a disposable 600-micron diode-laser fiber, without power, using mild pressure (Figure 3H-3J). Then, CSF was observed flowing freely through the opening with oscillatory movements of the margins of the ventriculostomy site (Figure 3K), confirming the successful ETV (Video). Through the opening on this membrane, we were able to pass the scope to access the prepontine and interpeduncular cistern and identify Liliequist membranes, with the basilar artery and the third cranial nerve (Figure 3L). The endoscope was then gently removed from the fetal head. Then, the uterus was closed and introduced back into the maternal sheep abdominal cavity.

Failures and Successful Fetal Endoscopic Third Ventriculostomy

Fetal endoscopic third ventriculostomy was successfully performed in 32/50 (67%) of the hydrocephalic fetal lambs and failure to achieve fenestration of the third ventricle happened in 18/50 (33%) cases for different causes.

In 11/18 cases (61%), failures were due to Bioglue material invading the lateral ventricles, blocking the FM and filling the third ventricle (Figure 4A). In other 5/18 cases (28%), failures were generated by an incorrect insertion or direction of the rigid endoscope into the lateral ventricle. Finally, 2/18 failures (11%) were due to impossibility to pass the scope through a very narrow FM as anatomic limitation.

FIGURE 4.: Failures in endoscopic fetal endoscopic third ventriculostomy. A, Blurred image with amber color of the cerebrospinal fluid because of Bioglue material invading the third or lateral ventricles. B, Presence of Bioglue polymer blocking the FM and filling the third ventricle making it impossible to pass the scope through a very narrow (anatomic limitation in sheep) FM. FM, foramen of Monro.

In 12/18 (67%), failures occurred on severe HCP cases and 6/18 (33%) on moderate HCP fetuses. In all cases without success in severe HCP with large ventricles, the cause of the failure was the caramel-like polymer blocking the scope navigation (Figure 4B); in 11/12 (92%) and the other cases were a narrow FM. In the failures due to wrong insertion of the scope or by an incorrect direction of the instrument, all 5/5 (100%) were in mild or moderate enlarged ventricles.

If we exclude the 11 cases where the polymer invaded the ventricular system making visualization or access to the third ventricle physically impossible, fetal endoscopic third ventriculostomy was successful in 32/39 cases, with a rate of 82% success (Table 3). Most of the successful cases were obtained in severe HCP where the space to navigate with the endoscope was wider.

TABLE 3. - Fetal ETV Successful Rates in HCP Sheep Model With Discarded Animal Model Issues

Groups	No. of fetus	Rate (%)	LVD		BPD		LVD/BPD
			E85 INJ	E105 ETV	E85 INJ	E105 ETV	E85 INJ	E105 ETV
Control	8		3.06 ± 0.19	3.56 ± 0.26	30.92 ± 1.48	45.56 ± 2.13	0.01 ± 0.004	0.07 ± 0.004
HCP + Unsuccessful ETV	7	17.95	3.10 ± 0.11	8.08 ± 2.93	33.94 ± 1.50	40.58 ± 6.08	0.09 ± 0.05	0.19 ± 0.03
Moderate HCP	5	23.81	3.10 ± 0.12	6.40 ± 0.45	33.82 ± 1.82	37.08 ± 0.22	0.09 ± 0.01	0.17 ± 0.01
Severe HCP	2	11.11	3.10 ± 0.14	12.30 ± 0.99	34.25 ± 0.35	49.35 ± 2.62	0.09 ± 0.01	0.25 ± 0.01
HCP + Successful FETV	32	82.05	2.95 ± 0.44	10.16 ± 3.34	34 ± 19 3.38	46.48 ± 5.36	0.087 ± 0.01	0.21 ± 0.06
Moderate HCP + FETV	16	76.19	2.93 ± 0.49	7.27 ± 1.07	33.66 ± 3.73	44.14 ± 6.63	0.09 ± 0.09	0.17 ± 0.03
Severe HCP + FETV	16	88.89	2.96 ± 0.41	13.10 ± 1.90	34.73 ± 3.01	48.50 ± 5.39	0.09 ± 0.01	0.27 ± 0.04

BPD, biparietal diameter; ETV, endoscopic third ventriculostomy; FETV, fetal endoscopic third ventriculostomy; HCP, hydrocephalus; INJ, injection; LVD, lateral ventricular diameter.

DISCUSSION

The standard surgical treatment of choice for congenital obstructive HCP is the postnatal CSF derivation achieved by either ventricular tube shunting or neuroendoscopic procedures to bypass the obstruction and restore the CSF flow. At that point, the decompression of the cerebral tissue usually arrives too late, as the critical period of fetal brain development was in-utero, and the tissue, cellular, and molecular consequences become irreversible compromising the motor and neurocognitive outcomes of these patients.⁶

In the past years, ETV has been gaining followers, and its use has become more universal, even for younger patients. The efficacy in newborns is controversial and on discussion, but it seems to work well for some specific etiologies such as aqueductal stenosis.¹⁴

Neonatal and fetal lamb models have a rich history in pediatric neurosurgery. In 1978, Di Rocco et al¹⁷ made some of the earliest observations about communicating HCP, determining a direct role of high-amplitude intraventricular CSF pulsations in the genesis of ventricular enlargement. Invasive neurosurgical fetal therapy began in the early 1980s when some pioneering groups introduced shunting procedures for the treatment of HCP.^7-13 After a period when large animal models were used, typically sheep and nonhuman primates, human application started.^13,15,18-23 After a period where large animal models, typically sheep and nonhuman primates, were used, human application started.^13,15,17-22

Clewel et al⁹ reported, in 1982, the clinical results of the first ventriculo-amniotic shunting in utero in human fetuses which, after a period of enthusiasm, finally revealed that these shunts failed to provide consistent ventricular decompression because of obstruction or migration issues.^9,10

In 2005, De Keersmaecker et al²⁴ reported that an experimental model was set up to assess the feasibility of neuroendoscopy in utero. Their animal model for HCP was created in fetal lambs by injecting blood into the lateral ventricle, simulating an intraventricular hemorrhage. They described and observed lateral ventricles with intrauterine ventriculoscopy as the first step in the development of considering intrauterine management options.

Our study is the first translational research project to assess the feasibility of fetal ETV in a sheep model of obstructive fetal HCP.¹⁶ We identified relevant differences between human and sheep intraventricular anatomy, especially the narrower FM and large interthalamic adhesion present in sheep. Moreover, the third ventricle appeared more compact and not as distensible in lambs as can be in humans.

We observed that ventricular dilatation was evident on prenatal imaging after BioGlue injections. Interestingly, we observed varying responses after BioGlue injections, with some fetal lambs developing severe or moderate HCP, mostly influenced by the volume of injected polymer. This fact allowed us to do a subanalysis on the feasibility of fetal ETV in both moderate and severe cases. Although the procedure was possible in both, we observed different difficulties of prenatal intervention. The main limitation for a successful fetal ETV was the diffusion of the BioGlue from the fourth ventricle through patent the Sylvius aqueduct to the lateral or third ventricles just before complete polymerization of the blend of the 2 components. It becomes very important to inject the polymer very slowly in the cisterna magna when creating the HCP or even reduce the dose to 1.2 mL to minimize these issues. The anatomic differences with humans are difficult to overcome, but at least the animal model seems more difficult than the human with a potential more favorable anatomy to achieve successful fetal ETV.

In most of the pediatric postnatal ETV, a neuroballoon is inflated to distent the third ventricle floor's fenestration, and many times this procedure is complemented with CP cauterization (CPC) in combination.²⁵ ETV combined with CPC has been used as a potential treatment in infants with spina bifida, posthemorrhagic HCP, congenital HCP, and postinfectious HCP.²⁵ This combination worked well in developing countries.²⁵ However, the results published have not been replicated in the United States. A multicenter study presented by the Hydrocephalus Research Society demonstrated similar results between ETV/CPC and ETV alone, questioning whether the CPC was necessary as a treatment for these specific patients.^26,27

Limitations

We consider that initially it is not required to use additional CPC in the fetal period, a fact that makes simpler and more feasible the prenatal intervention. We could consider adding these 2 technical additions in the future if the simplest technique demonstrates not to be fully effective. There remains the possibility to design specific neuroendoscopes for these purposes that could provide better performance in the fetal ETV intervention than the adapted current rigid ureteroscopes or fetoscopes, including flexible endoscopy as stated in some publications.²⁸ In the meantime, we consider both, this model for fetal severe HCP and this prenatal intervention, as a huge advance to develop new strategies and studies in the arrest of fetal HCP and as a powerful tool in the training for fetal neurosurgeons before jump to or during human clinical trials. Further investigations are required to define the efficacy and mechanisms of brain injury in obstructive HCP and to determine the therapeutic window for effective fetal intervention.

CONCLUSION

Despite anatomic differences between fetal lambs and humans, prenatal ETV is feasible in this induced fetal HCP ovine model. This intervention in the sheep model will be a great tool to develop more studies and help surgeons to train and develop a learning curve on their skills before starting clinical trials in humans. The next steps should demonstrate that the degree of brain mantle compression decreases significantly when this prenatal intervention is performed for severe or moderate fetal HCP in the fetal lamb.